De novo synthesis of bacteriochlorins

ABSTRACT

A method of making a bacteriochlorin is carried out by condensing a pair of compounds of Formula II 
     
       
         
         
             
             
         
       
     
     to produce the bacteriochlorin, wherein R is an acetal or aldehyde group. The condensing may be carried out in an organic solvent, preferably in the presence of an acid. The bacteriochlorins are useful for a variety of purposes such as active agents in photodynamic therapy, luminescent compounds in flow cytometry, solar cells, light harvesting arrays, and molecular memory devices.

RELATED APPLICATIONS

This application claims priority to and is a divisional of U.S. patentapplication Ser. No. 14/162,201, filed Jan. 23, 2014, now allowed, whichis a continuation of U.S. patent application Ser. No. 13/443,085, filedApr. 10, 2012, now U.S. Pat. No. 8,664,260, issued Mar. 4, 2014, whichis a continuation of U.S. patent application Ser. No. 12/413,903, filedMar. 30, 2009, now U.S. Pat. No. 8,173,692, issued May 8, 2012, which isa divisional of U.S. patent application Ser. No. 11/357,833, filed Feb.17, 2006, now U.S. Pat. No. 7,534,807, issued May 19, 2009, and alsoclaims the benefit of U.S. Provisional Applications Ser. Nos.60/654,270, filed Feb. 18, 2005, and 60/720,175, filed Sep. 23, 2005,the disclosures of which are incorporated by reference herein in theirentirety.

STATEMENT OF FEDERAL SUPPORT

This invention was made with government support under grant numberGM036238 awarded by the National Institutes of Health. The governmenthas certain rights to this invention.

FIELD OF THE INVENTION

The present invention concerns bacteriochlorins, methods andintermediates for the synthesis of bacteriochlorins, and methods ofusing such bacteriochlorins for, among other things, diagnostic andtherapeutic purposes such as photodynamic therapy (PDT), as luminescentcompounds in flow cytometry, in solar cells, in light harvesting arrays,and in molecular memory devices.

BACKGROUND OF THE INVENTION

The progressive 2e⁻/2H⁺ reduction of the porphyrinic macrocycle alongthe series porphyrin, chlorin (a dihydroporphyrin) and bacteriochlorin(a tetrahydroporphyrin) causes profound changes in chemical and physicalproperties (Scheme 1). The reduction alters the symmetry yet eachmacrocycle maintains an 18 π-electron conjugated system as required foraromaticity. One striking change upon reduction is the large increase inabsorption in the red or near-IR region of the spectrum.

The changes in physical properties have been famously exploited bybiological systems; the chlorin macrocycle provides the basis forchlorophyll a and b in plant photosynthesis while the bacteriochlorinmacrocycle provides the basis for bacteriochlorophyll a in bacterialphotosynthesis. The striking change in absorption is illustrated for arepresentative porphyrin, chlorin, and bacteriochlorin in FIG. 1.(Sternberg, E. D.; Dolphin, D. Tetrahedron 1998, 54, 4151-4202)

Two distinct types of bacteriochlorins occur in Nature,bacteriochlorophylls (type a, b, or g) and tolyporphins (A-J). Thebacteriochlorophylls serve as the principal light-absorbing pigments andenergy/electron-transfer components in bacterial photosynthetic systems.Bacteriochlorophyll a is the most widely distributed bacteriochlorinpigment and was the first bacteriochlorophyll isolated as a purecompound (Scheer, H. In The Porphyrins; Dolphin, D., Ed.; AcademicPress: New York, 1978; Vol. I, p 31; Scheer, H.; Inhoffen, H. H. In ThePorphyrins; Dolphin, D., Ed.; Academic Press: New York, 1978; Vol. I, p45). Tolyporphin A, a non-photosynthetic bacteriochlorin pigment, wasisolated from the cyanophyte microalga Tolypothrix nodosa in 1992, and anumber of additional tolyporphins that contain the bacteriochlorinsystem have since been isolated (Prinsep, M. R. et al., J. Am. Chem.Soc. 1992, 114, 385-387; Prinsep, M. R. et al., Tetrahedron 1995, 51,10523-10530; Prinsep, M. R. et al., J. Nat. Prod. 1998, 61, 1133-1136).The structures of bacteriochlorophyll a and tolyporphin A are shown inScheme II.

Surprisingly few methods exist for the preparation of bacteriochlorinsdespite the importance of this class of compounds (Johnson, A. W.;Oldfield, D. J. Chem. Soc. 1965, 4303-4312; Dinello, R. K.; Dolphin, D.J. Org. Chem. 1980, 45, 5196-5204; Chang, C. K.; Sotriou, C. J. Org.Chem. 1987, 52, 926-929; Kozyrev, A. N. et al., Tetrahedron Lett. 1996,37, 3781-3784; Shea, K. M. et al., Tetrahedron 2000, 56, 3139-3144).With regard to the naturally occurring bacteriochlorins, the totalsynthesis of the O,O-diacetate of tolyporphin A was reported severalyears ago by Kishi, entailing >20 steps and affording <5 mg of product(Wang, W.; Kishi, Y. Org. Lett. 1999, 1, 1129-1132). To our knowledge,no total syntheses of bacteriochlorophyll a have been reported. A chiefobstacle to handling bacteriochlorophyll a is its pronounced tendency toundergo dehydrogenation to give the corresponding chlorin. The sametendency for oxidative reversion to the chlorin or porphyrin occurs withbacteriochlorins that have been prepared by hydrogenation of theporphyrin or chlorin (Dorough, G. D.; Miller, J. R. J. Am. Chem. Soc.1952, 74, 6106-6108; Whitlock, H. W. et al., J. Am. Chem. Soc. 1969, 91,7485-7489; Fajer, J. et al., Proc. Nat. Acad. Sci. USA. 1974, 71,994-998; Bonnett, R. et al., Biochem. J. 1989, 261, 277-280; Grahn, M.F. et al., J. Photochem. Photobiol. B: Biol. 1997, 37, 261-266; Senge,M. O. et al., S. Tetrahedron 1998, 54, 3781-3798). More resilientbacteriochlorins have been prepared by derivatization of porphyrins orchlorins via vicinal dihydroxylation (typically followed by pinacolrearrangement for porphyrins that bear β-substituents) (Chang, C. K. etal., J. Chem. Soc. Chem. Commun. 1986, 1213-1215; Adams, K. R. et al.,J. Chem. Soc. Perkin Trans. 1 1992, 1465-1470; Pandey, R. K. et al.,Tetrahedron 1992, 51, 7815-7818; Kozyrev, A. N. et al., TetrahedronLett. 1996, 37, 3781-3784; Pandey, R. K. et al., J. Med. Chem. 1997, 40,2770-2779; Pandey, R. K. et al., J. Org. Chem. 1997, 62, 1463-1472;Zheng, G. et al., J. Org. Chem. 1999, 64, 3751-3754; Chen, Y. et al., J.Org. Chem. 2001, 66, 3930-3939; Li, G. et al., J. Org. Chem. 2004, 68,3762-3772), Diels-Alder reaction (Tomé, A. C. et al., Chem. Commun.1997, 1199-1200; Vincente, M. G. H. et al., Chem. Commun. 1998,2355-2356; Cavaleiro, J. A. S. et al., J. Hetetocyclic Chem. 2000, 37,527-534), or 1,3-dipolar cycloaddition (Silva, A. M. G. et al.,Tetrahedron Lett. 2002, 43, 603-605). While each of the derivatizationmethods has merit, a key limitation lies in the formation ofregioisomers upon use of porphyrinic substrates bearing a distinctpattern of substituents. On the other hand, modification of naturallyoccurring bacteriochlorophylls can yield elaborate bacteriochlorinderivatives, but the presence of a nearly full complement of peripheralsubstituents in the naturally available starting materials restrictssynthetic flexibility (Mironov, A. F et al., J. PorphyrinsPhthalocyanines 2003, 7, 725-730; Mironov, A. F. et al., Russ. J.Bioorg. Chem. 2003, 29, 190-197; Hartwich, G. et al., J. Am. Chem. Soc.1998, 120, 3675-3683; Tamiaki, H. et al., Tetrahedron: Asymmetry 1998,9, 2101-2111; Wasielewski, M. R. et al., J. Org. Chem. 1980, 45,1969-1974).

SUMMARY OF THE INVENTION

A first aspect of the present invention is a method of making a compoundof Formula I:

wherein:

X is selected from the group consisting of Se, NH, CH₂, O and S;

R¹, R², R³, R⁴, R⁵, R⁶ R⁷ and R⁸ is independently selected from thegroup consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl,arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido,cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, aryloxy,alkylthio, amino, alkylamino, arylalkylamino, disubstituted amino,acylamino, acyloxy, ester, amide, sulfoxyl, sulfonyl, sulfonate,sulfonic acid, sulfonamide, urea, alkoxylacylamino, aminoacyloxy,linking groups, and surface attachment groups;

or R¹ and R² together are ═O or spiroalkyl;

or R³ and R⁴ together are ═O or spiroalkyl;

and optionally but preferably subject to the proviso that (i) neither R¹nor R² is H, or (ii) neither R³ nor R⁴ is H; and

R⁸ is H, alkoxy or as given above;

the method comprising self-condensing a compound (or condensing a pairof compounds) of Formula II:

in an organic solvent in the presence of an acid to produce the compoundof Formula I, wherein:

R is an acetal or aldehyde group;

X and R¹ to R⁷ are as given above and R⁸ is H (or alkoxy, or asotherwise contributed by the acetal or aldehyde group); and

R¹¹ and R¹² are each H; or R¹¹ and R¹² together form a covalent bond.

Optionally, the compound can then be further derivatized to exchangehydrogen at the R⁸ position with the further substituents given above,in accordance with known techniques.

Compounds of the present invention (sometimes referred to as “activecompounds” herein) include compounds of Formula I, and pharmaceuticallyacceptable salts, prodrugs and conjugates thereof.

A further aspect of the invention is a method for treating a target in asubject in need thereof, comprising: (i) administering to the subjectthe active compound as described herein or a pharmaceutically acceptableconjugate thereof that preferentially associates with the target, and(ii) irradiating the target with light of a wavelength and intensitysufficient to treat the target. Suitable subjects include but are notlimited to subjects afflicted with opportunistic infections, with burns(particularly burns that have become infected), sepsis, with ulcers,periodontal disease, atherosclerosis, cosmetic and dermatologicconditions, acne, infectious diseases, tissues that require sealing suchas in wounds or surgical incisions, and subjects afflicted withneoplastic disease or cancer.

A further aspect of the invention is a photodynamic therapy method fortreating hyperproliferative tissue in a subject in need thereof,comprising: (i) administering to the subject an active compound asdescribed herein or a pharmaceutically acceptable conjugate thereof thatpreferentially associates with the hyperproliferative tissue, and (ii)irradiating the target with light of a wavelength and intensitysufficient to activate the compound, and thereby treat thehyperproliferative tissue.

A further aspect of the invention is a method for detecting the presenceof a hyperproliferative tissue in a subject, comprising: (i)administering to the subject an active compound as described herein or apharmaceutically acceptable conjugate thereof that preferentiallyassociates with the hyperproliferative tissue; and then (ii) visualizingthe compound within the patient.

A further aspect of the present invention is a kit to treathyperproliferative disorders, comprising the active compound describedherein or a pharmaceutically acceptable conjugate thereof andinstructions teaching a method of photodynamic therapy.

A further aspect of the present invention is a kit to label specifictissues for diagnosis comprising the active compound described herein ora pharmaceutically acceptable conjugate thereof and instructionsteaching a method of imaging (e.g., magnetic resonance imaging).

A further aspect of the present invention is, in a method of detectingparticles such as cells by flow cytometry, where the particles arelabelled with a detectable luminescent compound, the improvementcomprising utilizing a bacteriochlorin as described herein as theluminescent compound.

The foregoing and other objects and aspects of the invention areexplained in greater detail in the drawings herein and the specificationset forth below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Absorption spectra of Mg-octaethylporphyrin, chlorophyll a, andbacteriochlorophyll a. The molar absorption coefficients areε_(409 nm)=408,300 (Zass, E. et al., Helv. Chim. Acta 1980, 63,1048-1067), ε_(428.5 nm)=111,700 (Strain, H. H. et al., Biochim.Biophys. Acta 1963, 75, 306-311), and ε_(781 nm)=92,300 M⁻¹cm⁻¹(Connolly, J. S. et al., Photochem. Photobiol. 1982, 36, 565-574). Thespectra are normalized for comparison purposes.

FIG. 2. The effect of the different concentrations of BF₃.OEt₂ inbacteriochlorin formation (12+13). The reaction was carried out with 5mM of dihydrodipyrrin-acetal 11 in CH₃CN at room temperature. The yieldwas determined by absorption spectroscopy in CH₂Cl₂.

FIG. 3. The effect of different concentrations of dihydrodipyrrin-acetal11 on bacteriochlorin formation (12+13). The reaction was carried outwith BF₃.OEt₂ (10 equiv) in CH₃CN at room temperature. The yield wasdetermined by absorption spectroscopy in CH₂Cl₂.

FIG. 4A. Absorption spectra in toluene at room temperature of 12 and 13.

FIG. 4B. Emission Spectra in toluene at room temperature of 12 and 13.

The present invention is explained in greater detail in thespecification set forth below.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

“Acetal” as used herein refers to a group of the formula:

where R and R′ are each suitable groups, e.g., groups independentlyselected from the group consisting of alkyl, aryl, alkylaryl, or where Rand R′ together form a group —R″— where ═R″═ is an alkylene (i.e.,cycloalkyl). The acetal is preferably reasonably robust, and hence it ispreferred that at least one, or more preferably both, of R and R′ is notmethyl, and it is particularly preferred that neither R nor R′ is H.

“Aldehyde” as used herein refers to a group of the formula:

“Halo” as used herein refers to any suitable halogen, including —F, —Cl,—Br, and —I.

“Mercapto” as used herein refers to an —SH group.

“Azido” as used herein refers to an —N₃ group.

“Cyano” as used herein refers to a —CN group.

“Hydroxyl” as used herein refers to an —OH group.

“Nitro” as used herein refers to an —NO₂ group.

“Alkyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 10 carbonatoms. Representative examples of alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl,tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl,n-decyl, and the like. “Lower alkyl” as used herein, is a subset ofalkyl, in some embodiments preferred, and refers to a straight orbranched chain hydrocarbon group containing from 1 to 4 carbon atoms.Representative examples of lower alkyl include, but are not limited to,methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, andthe like. The term “akyl” or “loweralkyl” is intended to include bothsubstituted and unsubstituted alkyl or loweralkyl unless otherwiseindicated and these groups may be substituted with groups selected fromhalo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy(thereby creating a polyalkoxy such as polyethylene glycol), alkenyloxy,alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy,arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, mercapto,alkyl-S(O)_(m), haloalkyl-alkenyl-S(O)_(m), alkynyl-S(O)_(m),cycloalkyl-S(O)_(m), cycloalkylalkyl-S(O)_(m), aryl-S(O)_(m),arylalkyl-S(O)_(m), heterocyclo-S(O)_(m), heterocycloalkyl-S(O)_(m),amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino,cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino,heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino,acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy,nitro or cyano where m=0, 1, 2 or 3.

“Alkenyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 10 carbonatoms (or in loweralkenyl 1 to 4 carbon atoms) which include 1 to 4double bonds in the normal chain. Representative examples of alkenylinclude, but are not limited to, vinyl, 2-propenyl, 3-butenyl,2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene,and the like. The term “alkenyl” or “loweralkenyl” is intended toinclude both substituted and unsubstituted alkenyl or loweralkenylunless otherwise indicated and these groups may be substituted withgroups as described in connection with alkyl and loweralkyl above.

“Alkynyl” as used herein alone or as part of another group, refers to astraight or branched chain hydrocarbon containing from 1 to 10 carbonatoms (or in loweralkynyl 1 to 4 carbon atoms) which include 1 triplebond in the normal chain. Representative examples of alkynyl include,but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl,3-pentynyl, and the like. The term “alkynyl” or “loweralkynyl” isintended to include both substituted and unsubstituted alkynyl orloweralknynyl unless otherwise indicated and these groups may besubstituted with the same groups as set forth in connection with alkyland loweralkyl above.

“Alkoxy” as used herein alone or as part of another group, refers to analkyl or loweralkyl group, as defined herein (and thus includingsubstituted versions such as polyalkoxy), appended to the parentmolecular moiety through an oxy group, —O—. Representative examples ofalkoxy include, but are not limited to, methoxy, ethoxy, propoxy,2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy and the like.

“Acyl” as used herein alone or as part of another group refers to a—C(O)R radical, where R is any suitable substituent such as aryl, alkyl,alkenyl, alkynyl, cycloalkyl or other suitable substituent as describedherein.

“Haloalkyl” as used herein alone or as part of another group, refers toat least one halogen, as defined herein, appended to the parentmolecular moiety through an alkyl group, as defined herein.Representative examples of haloalkyl include, but are not limited to,chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl,2-chloro-3-fluoropentyl, and the like.

“Alkylthio” as used herein alone or as part of another group, refers toan alkyl group, as defined herein, appended to the parent molecularmoiety through a thio moiety, as defined herein. Representative examplesof alkylthio include, but are not limited, methylthio, ethylthio,tert-butylthio, hexylthio, and the like.

“Aryl” as used herein alone or as part of another group, refers to amonocyclic carbocyclic ring system or a bicyclic carbocyclic fused ringsystem having one or more aromatic rings. Representative examples ofaryl include, azulenyl, indanyl, indenyl, naphthyl, phenyl,tetrahydronaphthyl, and the like. The term “aryl” is intended to includeboth substituted and unsubstituted aryl unless otherwise indicated andthese groups may be substituted with the same groups as set forth inconnection with alkyl and loweralkyl above.

“Arylalkyl” as used herein alone or as part of another group, refers toan aryl group, as defined herein, appended to the parent molecularmoiety through an alkyl group, as defined herein. Representativeexamples of arylalkyl include, but are not limited to, benzyl,2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

“Amino” as used herein means the radical —NH₂.

“Alkylamino” as used herein alone or as part of another group means theradical —NHR, where R is an alkyl group.

“Arylalkylamino” as used herein alone or as part of another group meansthe radical —NHR, where R is an arylalkyl group.

“Disubstituted-amino” as used herein alone or as part of another groupmeans the radical —NR_(a)R_(b), where R_(a) and R_(b) are independentlyselected from the groups alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl.

“Acylamino” as used herein alone or as part of another group means theradical —NR_(a)R_(b), where R_(a) is an acyl group as defined herein andR_(b) is selected from the groups hydrogen, alkyl, haloalkyl, alkenyl,alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo,heterocycloalkyl.

“Acyloxy” as used herein alone or as part of another group means theradical —OR, where R is an acyl group as defined herein.

“Ester” as used herein alone or as part of another group refers to a—C(O)OR radical, where R is any suitable substituent such as alkyl,cycloalkyl, alkenyl, alkynyl or aryl.

“Formyl” as used herein refers to a —C(O)H group.

“Carboxylic acid” as used herein refers to a —C(O)OH group.

“Sulfoxyl” as used herein refers to a compound of the formula —S(O)R,where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl,alkynyl or aryl.

“Sulfonyl as used herein refers to a compound of the formula —S(O)(O)R,where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl,alkynyl or aryl.

“Sulfonate” as used herein refers to a compound of the formula—S(O)(O)OR, where R is any suitable substituent such as alkyl,cycloalkyl, alkenyl, alkynyl or aryl.

“Sulfonic acid as used herein refers to a compound of the formula—S(O)(O)OH.

“Amide” as used herein alone or as part of another group refers to a—C(O)NR_(a)R_(b) radical, where R_(a) and R_(b) are any suitablesubstituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Sulfonamide” as used herein alone or as part of another group refers toan —S(O)₂NR_(a)R_(b) radical, where R_(a) and R_(b) are any suitablesubstituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Urea” as used herein alone or as part of another group refers to an—N(R_(c))C(O)NR_(a)R_(b) radical, where R_(a), R_(b) and R_(c) are anysuitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl oraryl.

“Alkoxyacylamino” as used herein alone or as part of another grouprefers to an —N(R_(a))C(O)OR_(b) radical, where R_(a), R_(b) are anysuitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl oraryl.

“Aminoacyloxy” as used herein alone or as part of another group refersto an —OC(O)NR_(a)R_(b) radical, where R_(a) and R_(b) are any suitablesubstituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

“Cycloalkyl” as used herein alone or as part of another group, refers toa saturated or partially unsaturated cyclic hydrocarbon group containingfrom 3, 4 or 5 to 6, 7 or 8 carbons (which carbons may be replaced in aheterocyclic group as discussed below). Representative examples ofcycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. These rings may be optionally substitutedwith additional substituents as described herein such as halo orloweralkyl. The term “cycloalkyl” is generic and intended to includeheterocyclic groups as discussed below unless specified otherwise.

“Heterocyclic group” or “heterocyclo” as used herein alone or as part ofanother group, refers to an aliphatic (e.g., fully or partiallysaturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or abicyclic-ring system. Monocyclic ring systems are exemplified by any 5or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independentlyselected from oxygen, nitrogen and sulfur. The 5 membered ring has from0-2 double bonds and the 6 membered ring has from 0-3 double bonds.Representative examples of monocyclic ring systems include, but are notlimited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane,dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine,isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline,isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine,oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran,pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine,pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran,tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline,thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene,thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole,trithiane, and the like. Bicyclic ring systems are exemplified by any ofthe above monocyclic ring systems fused to an aryl group as definedherein, a cycloalkyl group as defined herein, or another monocyclic ringsystem as defined herein. Representative examples of bicyclic ringsystems include but are not limited to, for example, benzimidazole,benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole,benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine,1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine,naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline,isoquinoline, phthalazine, purine, pyranopyridine, quinoline,quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline,tetrahydroquinoline, thiopyranopyridine, and the like. These ringsinclude quaternized derivatives thereof and may be optionallysubstituted with groups selected from halo, alkyl, haloalkyl, alkenyl,alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo,heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy,cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy,heterocyclolalkyloxy, mercapto, alkyl-S(O)_(m), haloalkyl-S(O)_(m),alkenyl-S(O)_(m), alkynyl-S(O)_(m), cycloalkyl-S(O)_(m),cycloalkylalkyl-S(O)_(m), aryl-S(O)_(m), arylalkyl-S(O)_(m),heterocyclo-S(O)_(m), heterocycloalkyl-S(O)_(m), amino, alkylamino,alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino,cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino,heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester,amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyanowhere m=0, 1, 2 or 3. Preferred heterocyclo groups include pyridyl andimidazolyl groups, these terms including the the quaternized derivativesthereof, including but not limited to quaternary pyridyl and imidazolylgroups, examples of which include but are not limited to:

where R and R′ are each a suitable substitutent as described inconnection with “alkyl” above, and particularly alkyl (such as methyl,ethyl or propyl), arylalkyl (such as benzyl), optionally substitutedwith hydroxy (—OH), phosphonic acid (—PO₃H₂) or sulfonic acid (—SO₃H),and X⁻ is a counterion.

“Spiroalkyl” as used herein alone or as part of another group, refers toa straight or branched chain hydrocarbon, saturated or unsaturated,containing from 3 to 8 carbon atoms. Representative examples include,but are not limited to, —CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂—, —CH₂CH₂CH₂CH₂CH₂—,—CH₂CH₂CHCHCH₂—, —CH₂CH₂CH₂CH₂CH₂CH₂—, etc. The term “spiroalkyl” isintended to include both substituted and unsubstituted “spiroalkyl”unless otherwise indicated and these groups may be substituted withgroups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo,heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy,cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy,heterocyclolalkyloxy, mercapto, alkyl-S(O)_(m), haloalkyl-S(O)_(m),alkenyl-S(O)_(m), alkynyl-S(O)_(m), cycloalkyl-S(O)_(m),cycloalkylalkyl-S(O)_(m), aryl-S(O)_(m), arylalkyl-S(O)_(m),heterocyclo-S(O)_(m), heterocycloalkyl-S(O)_(m), amino, alkylamino,alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino,cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclo amino,heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester,amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyanowhere m=0, 1 or 2.

“Treatment” as used herein means any manner in which one or more of thesymptoms of a disease or disorder are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein, such as use for treating hyperproliferatingtissue or neovascularization mediated diseases or disorders, or diseasesor disorders in which hyperproliferating tissue or neovascularization isimplicated. As used herein, amelioration of the symptoms of a particulardisorder by administration of a particular compound or pharmaceuticalcomposition refers to any lessening, whether permanent or temporary,lasting or transient that can be attributed to or associated withadministration of the composition.

“Prodrug” as used herein is a compound that, upon in vivoadministration, is metabolized by one or more steps or processes orotherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound.

“Antibody” as used herein refers generally to immunoglobulins orfragments thereof that specifically bind to antigens to form immunecomplexes. The antibody may be whole immunoglobulin of any class, e.g.,IgG, IgM, IgA, IgD, IgE, chimeric or hybrid antibodies with dual ormultiple antigen or epitope specificities. It can be a polyclonalantibody, preferably an affinity-purified antibody from a human or anappropriate animal, e.g., a primate, goat, rabbit, mouse or the like.Monoclonal antibodies are also suitable for use in the presentinvention, and are preferred because of their high specificities. Theyare readily prepared by what are now considered conventional proceduresof immunization of mammals with immunogenic antigen preparation, fusionof immune lymph or spleen cells with an immortal myeloma cell line, andisolation of specific hybridoma clones. More unconventional methods ofpreparing monoclonal antibodies are not excluded, such as interspeciesfusions and genetic engineering manipulations of hypervariable regions,since it is primarily the antigen specificity of the antibodies thataffects their utility. Newer techniques for production of monoclonalscan also be used, e.g., human monoclonals, interspecies monoclonals,chimeric (e.g., human/mouse) monoclonals, genetically engineeredantibodies and the like.

“Infecting agent” as used herein denotes invading microbes or parasites.As used herein, “microbe” denotes virus, bacteria, rickettsia,mycoplasma, protozoa, fungi and like microorganisms, and “parasite”denotes infectious, generally microscopic or very small multicellularinvertebrates, or ova or juvenile forms thereof, which are susceptibleto antibody-induced clearance or lytic or phagocytic destruction, e.g.,malarial parasites, spirochetes and the like.

“Tumor” as used herein denotes a neoplasm, and includes both benign andmalignant tumors. This term particularly includes malignant tumors whichcan be either solid (such as a breast, liver, or prostate carcinoma) ornon-solid (such as a leukemia). Tumors can also be further divided intosubtypes, such as adenocarcinomas (e.g. of the breast, prostate orlung).

“Target” as used herein denotes the object that is intended to bedetected, diagnosed, impaired or destroyed by the methods providedherein, and includes target cells, target tissues, and targetcompositions. “Target tissues” and “target cells” as used herein arethose tissues that are intended to be impaired or destroyed by thistreatment method. Photosensitizing compounds bind to or collect in thesetarget tissues or target cells; then when sufficient radiation isapplied, these tissues or cells are impaired or destroyed. Target cellsare cells in target tissue, and the target tissue includes, but is notlimited to, vascular endothelial tissue, abnormal vascular walls oftumors, solid tumors such as (but not limited to) tumors of the head andneck, tumors of the eye, tumors of the gastrointestinal tract, tumors ofthe liver, tumors of the breast, tumors of the prostate, tumors of thelung, nonsolid tumors and malignant cells of the hematopoietic andlymphoid tissue, neovascular tissue, other lesions in the vascularsystem, bone marrow, and tissue or cells related to autoimmune disease.Also included among target cells are cells undergoing substantially morerapid division as compared to non-target cells.

“Non-target tissues” as used herein are all the tissues of the subjectwhich are not intended to be impaired or destroyed by the treatmentmethod. These non-target tissues include but are not limited to healthyblood cells, and other normal tissue, not otherwise identified to betargeted.

“Target compositions” as used herein are those compositions that areintended to be impaired or destroyed by this treatment method, and mayinclude one or more pathogenic agents, including but not limited tobacteria, viruses, fungi, protozoa, and toxins as well as cells andtissues infected or infiltrated therewith. The term “targetcompositions” also includes, but is not limited to, infectious organicparticles such as prions, toxins, peptides, polymers, and othercompounds that may be selectively and specifically identified as anorganic target that is intended to be impaired or destroyed by thistreatment method.

“Hyperproliferative tissue” as used herein means tissue that grows outof control and includes neoplastic tissue, tumors and unbridled vesselgrowth such as blood vessel growth found in age-related maculardegeneration and often occurring after glaucoma surgeries.

“Hyperproliferative disorders” as used herein denotes those conditionsdisorders sharing as an underlying pathology excessive cellproliferation caused by unregulated or abnormal cell growth, and includeuncontrolled angiogenesis. Examples of such hyperproliferative disordersinclude, but are not limited to, cancers or carcinomas, acute andmembrano-proliferative glomerulonephritis, myelomas, psoriasis,atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabeticretinopathies, macular degeneration, corneal neovascularization,choroidal hemangioma, recurrence of pterygii, and scarring from excimerlaser surgery and glaucoma filtering surgery.

“Therapeutically effective dose” as used herein is a dose sufficient toprevent advancement, or to cause regression of the disease, or which iscapable of relieving symptoms caused by the disease.

“Irradiating” and “irradiation” as used herein includes exposing asubject to all wavelengths of light. Preferably, the irradiatingwavelength is selected to match the wavelength(s) which excite thephotosensitive compound. Preferably, the radiation wavelength matchesthe excitation wavelength of the photosensitive compound and has lowabsorption by the non-target tissues of the subject, including bloodproteins.

“Biological materials” as used herein refers to both tissues (such asbiopsy tissues) and cells, as well as biological fluids such as blood,urine, plasma, cerebrospinal fluid, mucus, sputum, etc.

Irradiation is further defined herein by its coherence (laser) ornon-coherence (non-laser), as well as intensity, duration, and timingwith respect to dosing using the photosensitizing compound. Theintensity or fluence rate must be sufficient for the light to reach thetarget tissue. The duration or total fluence dose must be sufficient tophotoactivate enough photosensitizing compound to act on the targettissue. Timing with respect to dosing with the photosensitizing compoundis important, because 1) the administered photosensitizing compoundrequires some time to home in on target tissue and 2) the blood level ofmany photosensitizing compounds decreases with time. The radiationenergy is provided by an energy source, such as a laser or cold cathodelight source, that is external to the subject, or that is implanted inthe subject, or that is introduced into a subject, such as by acatheter, optical fiber or by ingesting the light source in capsule orpill form (e.g., as disclosed in. U.S. Pat. No. 6,273,904 (2001)).

While one preferred embodiment of the present invention is drawn to theuse of light energy for administering photodynamic therapy (PDT) todestroy tumors, other forms of energy are within the scope of thisinvention, as will be understood by those of ordinary skill in the art.Such forms of energy include, but are not limited to: thermal, sonic,ultrasonic, chemical, light, microwave, ionizing (such as x-ray andgamma ray), mechanical, and electrical. For example, sonodynamicallyinduced or activated agents include, but are not limited to:gallium-porphyrin complex (see Yumita et al., Cancer Letters 112: 79-86(1997)), other porphyrin complexes, such as protoporphyrin andhematoporphyrin (see Umemura et al., Ultrasonics Sonochemistry 3:S187-S191 (1996)); other cancer drugs, such as daunorubicin andadriamycin, used in the presence of ultrasound therapy (see Yumita etal., Japan J. Hyperthermic Oncology 3(2):175-182 (1987)).

“Coupling agent” as used herein, refers to a reagent capable of couplinga photosensitizer to a targeting agent

“Targeting agent” refers to a compound that homes in on orpreferentially associates or binds to a particular tissue, receptor,infecting agent or other area of the body of the subject to be treated,such as a target tissue or target composition. Examples of a targetingagent include but are not limited to an antibody, a ligand, one memberof a ligand-receptor binding pair, nucleic acids, proteins and peptides,and liposomal suspensions, including tissue-targeted liposomes.

“Specific binding pair” and “ligand-receptor binding pair” as usedherein refers to two different molecules, where one of the molecules hasan area on the surface or in a cavity which specifically attracts orbinds to a particular spatial or polar organization of the othermolecule, causing both molecules to have an affinity for each other. Themembers of the specific binding pair are referred to as ligand andreceptor (anti-ligand). The terms ligand and receptor are intended toencompass the entire ligand or receptor or portions thereof sufficientfor binding to occur between the ligand and the receptor. Examples ofligand-receptor binding pairs include, but are not limited to, hormonesand hormone receptors, for example epidermal growth factor and epidermalgrowth factor receptor, tumor necrosis factor-.alpha. and tumor necrosisfactor-receptor, and interferon and interferon receptor; avidin andbiotin or antibiotin; antibody and antigen pairs; enzymes andsubstrates, drug and drug receptor; cell-surface antigen and lectin; twocomplementary nucleic acid strands; nucleic acid strands andcomplementary oligonucleotides; interleukin and interleukin receptor;and stimulating factors and their receptors, such asgranulocyte-macrophage colony stimulating factor (GMCSF) and GMCSFreceptor and macrophage colony stimulating factor (MCSF) and MCSFreceptor.

“Linkers” are aromatic or aliphatic groups (which may be substituted orunsubstituted and may optionally contain heteroatoms such as N, O, or S)that are utilized to couple a bioconjugatable group, cross-couplinggroup, surface attachment group, hydrophilic group or the like to theparent molecule. Examples include but are not limited to aryl, alkyl,heteroaryl, heteroalkyl (e.g., oligoethylene glycol), peptide, andpolysaccharide linkers, etc.

Subjects to be treated by the methods of the present invention fordiagnostic or therapeutic purposes include both human subjects andanimal subjects (particularly mammalian subjects such as dogs, cats,horses, monkeys, chimpanzees, etc.) for veterinary purposes.

The disclosures of all United States Patent references cited herein areto be incorporated by reference herein as if fully set forth.

1. Compounds and Methods of Making.

As noted above, an aspect of the present invention is a method of makinga compound of Formula I:

wherein:

X is selected from the group consisting of Se, NH, CH₂, O and S;

R¹, R², R³, R⁴, R⁵, R⁶ R⁷ and R⁸ are each independently selected fromthe group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, heterocyclo,heterocycloalkyl, heterocycloalkenyl, heterocycloalkynyl, aryl,arylalkyl, arylalkenyl, arylalkynyl, heteroaryl, heteroarylalkyl,heteroarylalkenyl, heteroarylalkynyl, alkoxy, halo, mercapto, azido,cyano, formyl, carboxylic acid, hydroxyl, nitro, acyl, alkylthio, amino,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,ester, amide, sulfoxyl, sulfonyl, sulfonate, sulfonic acid, sulfonamide,urea, alkoxylacylamino, aminoacyloxy, linking groups, and surfaceattachment groups;

or R¹ and R² together are ═O or spiroalkyl (in which case preferablyneither R³ nor R⁴ is H);

or R³ and R⁴ together are ═O or spiroalkyl (in which case preferablyneither R¹ nor R² is H);

optionally but preferably subject to the proviso that (i) neither R¹ norR² is H, or (ii) neither R³ nor R⁴ is H; and

R⁸ is H or as given above;

the method comprising self-condensing a compound (or condensing a pairof compounds) of Formula II:

in an organic solvent in the presence of an acid to produce the compoundof Formula I, wherein:

R is an acetal group;

X and R¹ to R⁷ are as given above and R⁸ is H; and

R¹¹ and R¹² are each H; or R¹¹ and R¹² together form a covalent bond.

Optionally, R⁸ is further substituted to replace H with additionalsubstituents as described above in accordance with known techniques.

It will be appreciated that when a single compound is self-condensed,the various groups R¹ through R⁷ will be symmetric in the compounds ofFormula I, but that when a pair of compounds with different patterns ofsubstituents are condensed, the various groups R¹ through R⁷ may beunsymmetric or different in the compounds of Formula I).

In some embodiments, R¹, R², R³, and R⁴ are preferably eachindependently selected from the group consisting of H, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl,cycloalkylalkynyl, heterocyclo, heterocycloalkyl, heterocycloalkenyl,heterocycloalkynyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkoxy,carboxylic acid, hydroxyl, alkylthio, amino, alkylamino, arylalkylamino,disubstituted amino, acylamino, acyloxy, ester, In some embodiments, R¹,R², R³, and R⁴ are most preferably each independently selected from thegroup consisting of H and alkyl.

In some embodiments, preferably, R¹ and R² are each independentlyselected from the group consisting of H, alkyl, cycloalkyl, aryl,alkoxy, halo, mercapto, hydroxyl, nitro, acyl, alkoxy, alkylthio, amino,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,amide, sulfonamide, urea, alkoxylacylamino, aminoacyloxy, and linkinggroups. Most preferably R¹ and R² are each independently selected fromthe group consisting of H, alkyl, aryl, alkoxy, halo, mercapto, cyano,hydroxyl, nitro, acyl, alkoxy, alkylthio, alkylamino, acyloxy, amide,and linking groups. In some embodiments R¹ and R² are preferably not H,alkyl or cycloalkyl (“cycloalkyl” including heterocyclo), particularlynot alkyl or cycloalkyl, and most particularly one is not alkyl when theother is cycloalkyl.

In some embodiments, preferably, R³ and R⁴ are each independentlyselected from the group consisting of alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, aryl,arylalkyl, arylalkenyl, arylalkynyl, and linking groups. Mostpreferably, R³ and R⁴ are each independently selected from the groupconsisting of alkyl, cycloalkyl, aryl, arylalkyl, and linking groups.

In some embodiments, preferably, R⁵ is selected from the groupconsisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,cycloalkylalkenyl, cycloalkylalkynyl, aryl, arylalkyl, arylalkenyl,arylalkynyl, alkoxy, halo, cyano, nitro, acyl, alkoxy, alkylthio, amino,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,amide, sulfonamide, urea, alkoxylacylamino, aminoacyloxy, and linkinggroups. Most preferably, R⁵ is selected from the group consisting of H,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl,arylalkynyl, alkoxy, halo, cyano, nitro, acyl, alkoxy, alkylthio,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,amide, and linking groups.

In some embodiments R⁵ is preferably not H or alkyl, and particularlynot H.

In some embodiments, preferably, R⁶ and R⁷ are each independentlyselected from the group consisting of H, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkylalkyl, cycloalkylalkenyl, cycloalkylalkynyl, aryl,arylalkyl, arylalkenyl, arylalkynyl, alkoxy, halo, cyano, nitro, acyl,alkoxy, alkylthio, amino, alkylamino, arylalkylamino, disubstitutedamino, acylamino, acyloxy, amide, sulfonamide, urea, alkoxylacylamino,aminoacyloxy, and linking groups. Most preferably, R⁶ and R⁷ are eachindependently selected from the group consisting of H, alkyl, alkenyl,alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkoxy,halo, cyano, nitro, acyl, alkoxy, alkylthio, alkylamino, arylalkylamino,disubstituted amino, acylamino, acyloxy, amide, and linking groups.

In some embodiments at least one or both R⁶ is preferably neither H noralkyl, and particularly not H.

In some embodiments at least one or both R⁷ is preferably neither H noralkyl, and particularly not methyl.

In some embodiments, preferably, R⁸ is selected from the groupconsisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,cycloalkylalkenyl, cycloalkylalkynyl, aryl, arylalkyl, arylalkenyl,arylalkynyl, alkoxy, halo, cyano, nitro, acyl, alkoxy, alkylthio, amino,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,amide, sulfonamide, urea, alkoxylacylamino, aminoacyloxy, and linkinggroups. Most preferably, R⁸ is selected from the group consisting of H,alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl,arylalkynyl, alkoxy, halo, cyano, nitro, acyl, alkoxy, alkylthio,alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy,amide, and linking groups.

In some embodiments R⁸ is preferably not H or alkyl, and particularlynot H.

In some embodiments compounds of Formula I are subject to the provisothat, when X is NH: R¹ is not cycloalkyl; or R² is not methyl; or R⁵ isnot H; or R⁶ is not H; or R⁷ is not methyl.

Synthesis via acetal intermediates. Compounds of Formula I are made fromcompounds of Formula IIa or IIb as shown below by treating the compoundsof Formulas IIa or IIb with an acid in an organic solvent. The acid isnot critical, with examples including but not limited to BF₃ etherate,SnCl₄, InCl₃, trifluoroacetic acid, and toluenesulfonic acid. Theorganic solvent is not critical with examples including but not limitedto acetonitrile, methylene chloride, chloroform, tetrahydrofuran,chlorobenzene, ethanol, and combinations thereof. The reaction may becarried out at any suitable temperature, such as 0 to 100° C., andconveniently at room temperature, for any suitable time period, such asfor a few minutes, 1 to 4 hours, or a day. The reaction mixture ispreferably nonaqueous but need not be anhydrous, and may conveniently becarried out exposed to air. When compounds of Formula IIb are utilizedthe reaction mixture preferably includes an oxidizing agent such as airor DDQ.

in Formulas IIa and IIb, R¹ through R⁷ are the same as given above inconnection with Formula I, and R is acetal.

Compounds of Formulas IIa and IIb are made from compounds of FormulaIII.

R¹ through R⁷ are the same as given above in connection with Formula I,and R is acetal. In general, compounds of Formula IIa are produced bydeprotonating a compound of Formula III (e.g., by treating withanhydrous sodium methoxide) to produce a nitronate anion intermediate,and then cyclizing the intermediate with a deoxygenating agent (e.g., bycombining the intermediate with an aqueous buffered TiCl₃ solution) toproduce the compound of Formula IIa. Reaction conditions are notcritical and numerous variations will be apparent to those skilled inthe art. In general, compounds of Formula IIb are produced by treating acompound of Formula III with a metal (e.g., zinc and acetic acid inethanol) to produce an N-oxide intermediate, and then cyclizing theintermediate with a deoxygenating agent (e.g., Ti(0), Zn, NaOH/methanol;Zn, aqueous NH₄Cl/THF; FeSO₄, aqueous NH₄Cl/CH₃CN; Mg or Fe,AcONH₄/methanol; Ph₃P/toluene; S/toluene; NaN₃/toluene; Zn, NaI,Me₃SiCl/CH₃CN; etc.) to produce the compound of Formula IIb. Againreaction conditions are not critical and numerous variations will beapparent to those skilled in the art.

Synthesis via aldehyde intermediates. Compounds of Formula I are madefrom compounds of Formula IIa or IIb as shown above, where R is analdehyde, by treating the compounds of Formulas IIa or IIb with an acidin an organic solvent in like manner as described above. Compounds ofFormula IIa or IIb where R is an aldehyde are made by oxidizing acorresponding compound of Formula V:

in an organic solvent in the presence of an oxidizing agent to producethe compound of Formula II. Any suitable solvent can be used,particularly ethereal solvents such as 1,4-dioxane, tetrahydrofuran,diethyl ether and dimethoxyethane. The reaction conditions are notcritical and the reaction may be carried out at any suitabletemperature, for example 0 to 100° C., preferably room temperature, forany suitable time, typically one to two hours. SeO₂ is currentlypreferred as the oxidizing agent, but any suitable oxidizing agent maybe used. In general, when relatively powerful oxidizing agents areemployed with alkyl groups that are activated by the presence of a itbond (allylic), the alkyl group can be oxidized to the aldehyde orketone. The most common reagents for these transformations are seleniumdioxide (SeO₂), chromium trioxide (CrO₃), chromyl chloride (CrO₂Cl₂),and Pb(OAc)₄. In addition, t-BuOOH/CuI oxidizes the allylic carbon ofalkenyl conjugated ketones (Organic Synthesis, 2^(nd) Ed; Smith, M. B.;McGraw-Hill Higher Education: 2002; 272-279) and can also be used asoxidizing agents herein. A variety of chromium reagents have been usedfor allylic oxidations ((a) Dauben, W. G.; Lorber, M.; Fullerton, D. S.J. Org. Chem. 1969, 34, 3587-3592. (b) Fullerton, D. S.; Chen, C. M.Synth. Commun. 1976, 6, 217-220. (c) Salmond, W. G.; Barta, M. A.;Havens, J. L. J. Org. Chem. 1978, 43, 2057-2059. (d) Parish, E. J.;Chitrakorn, S.; Wei, T.-Y. Synth. Commun. 1986, 16, 1371-1375. (e)Parish, E. J.; Wei, T.-Y. Synth. Commun. 1987, 17, 1227-1233. (f)Marshall, C. W.; Ray, R. E.; Laos, I.; Riegel, B. J. Am. Chem. Soc.1975, 79, 6308-6313. (g) Amann, A.; Ourisson, G.; Luu, B. Synthesis1987, 1002-1005. (h) Bora, U.; Chaudhuri, M. K.; Dey, D.; Kalita, D.;Kharmawphlang, W.; Mandal, G. C. Tetrahedron 2001, 57, 2445-2448) andcan also be used as oxidizing agents herein. Examples includeCrO₃-pyridine complex, CrO₃ and 3,5-dimethylpyrazole, pyridiniumchlorochromate (PCC), pyridinium dichromate (PDC), sodium chromate,sodium dichromate in acetic acid, pyridinium fluorochromate, and3,5-dimethylpyrazolium fluorochromate (VI). The 5-methyl group of apyrrole-2-ester was oxidized by ceric ammonium nitrate ((a) Huggins, M.T.; Lightner, D. A. Tetrahedron 2000, 56, 1797-1810. (b) Tipton, A. K.;Lightner, D. A.; McDonagh, A. F. J. Org. Chem. 2001, 66, 1832-1838) andthis can also be used as an oxidizing agent herein.

Compounds of Formula I may be produced wherein R⁸ is H by the methodsdescribed above, and then R⁸ brominated in accordance with knowntechniques and further substituents added at position R⁸ in accordancewith known techniques. Likewise other substituents can be added atpositions R¹ through R⁷ by substitution (e.g., by bromination orformylation) in accordance with known techniques.

Compounds of Formula I may be metalated with any suitable metal inaccordance with known techniques. See, e.g., U.S. Pat. No. 6,208,553.Suitable metals include but are not limited to Pd(II), Pt(II), Mg(II),Zn(II), Al(III), Ga(III), In(III), Sn(IV), Cu(II), Ni(II), and Au(III).Where the metal is trivalent or tetravalent a counterion is included asnecessary in accordance with known techniques.

Linking groups for conjugates. Linking groups are included in compoundsof Formula I to provide a reactive site for conjugation so that thecompounds may be coupled to or conjugated to other groups such asproteins, peptides, targeting agents such as antibodies, polymers,particles such as nanoparticles, organic, polymeric or inorganic beads,other solid support surfaces, etc., to form additional active compoundsof the invention. In general each group is attached to a linking groupincluding a linker which can be aryl, alkyl, heteroaryl, heteroalkyl(e.g., oligoethylene glycol), peptide, polysaccharide, etc. The linkinggroup may be simply a reactive attachment group or moiety (e.g., —R′where R′ is a reactive group such as bromo), or may comprise acombination of an intervening group coupled to a reactive group (e.g.,—R″R′, where R′ is a reactive group and R′ is an intervening group suchas a hydrophilic group).

For bioconjugation purposes, the choice of water-solubilizing group(s)and conjugation groups is made so as to achieve orthogonal coupling. Forexample, if a carboxylic acid is used for water solubility, an aldehydemight be used for bioconjugation (via reductive amination with anamino-substituted biomolecule). If a carboxylic acid is used forbioconjugation (via carbodiimide-activation and coupling with anamino-substituted biomolecule), then a complementary group can be usedfor water solubility (e.g., sulfonic acid, guanidinium, pyridinium).Bioconjugatable groups include amines (including amine derivatives) suchas isocyanates, isothiocyanates, iodoacetamides, azides, diazoniumsalts, etc. acids or acid derivatives such as N-hydroxysuccinimideesters (more generally, active esters derived from carboxylic acids;e.g., p-nitrophenyl ester), acid hydrazides, etc., and other linkinggroups such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides,epoxides, hydroxyl groups, thiol groups, maleimides, aziridines,acryloyls, halo groups, biotin, 2-Iminobiotin, etc. Linking groups suchas the foregoing are known and described in U.S. Pat. Nos. 6,728,129;6,657,884; 6,212,093; and 6,208,553.

Conjugates. Other groups can be attached to the bacteriochlorin to forma conjugate by means of a linking group to tune or adjust the solubilityproperties of the bacteriochlorin, including hydrophobic groups,hydrophilic groups, polar groups, or amphipathic groups. The polargroups include carboxylic acid, sulfonic acid, guanidinium,carbohydrate, hydroxy, amino acid, pyridinium, imidazolium, etc. Suchgroups can be attached to substituents that are linear or branched alkyl(e.g., swallowtail), aryl, heteroaryl, heteroalkyl (e.g., oligoethyleneglycol), peptide, polysaccharide, etc. Targeting groups such asantibodies, proteins, peptides, and nucleic acids may be attached bymeans of the linking group. Particles such as nanoparticles, glassbeads, etc. may be attached by means of the linking group. Where suchadditional compounds are attached to form a conjugate that may beattached directly to the bacteriochlorin or attached by means of anintervening group such as a hydrophilic group, depending upon theparticular linking group employed (as noted above).

Hydrophilic groups. Compounds of the present invention may includehydrophilic groups coupled at the linking sites noted above, e.g.,covalently coupled thereto, to facilitate delivery thereof, or improvestability, in accordance with known techniques (e.g., to the N-terminusof the peptide). Suitable hydrophilic groups are typically polyols orpolyalkylene oxide groups, including straight and branched-chainpolyols, with particularly examples including but not limited topoly(propylene glycol), polyethylene-polypropylene glycol orpoly(ethylene glycol). The hydrophilic groups may have a number averagemolecular weight of 20,000 to 40,000 or 60,000. Suitable hydrophilicgroups and the manner of coupling thereof are known and described in,for example, U.S. Pat. Nos. 4,179,337; 5,681,811; 6,524,570; 6,656,906;6,716,811; and 6,720,306. For example, compounds can be pegylated usinga single 40,000 molecular weight polyethylene glycol moiety that isattached to the compound by means of a linking group.

Surface attachment groups. As noted above, compounds of the inventioncan be substituted with a surface attachment group, which may be inprotected or unprotected form. A surface attachment group may be areactive group coupled directly to the bacteriochlorin, or coupled tothe bacteriochlorin by means of an intervening linker. Linkers L can bearyl, alkyl, heteroaryl, heteroalkyl (e.g., oligoethylene glycol),peptide, polysaccharide, etc. Examples of surface attachment groups(with the reactive site or group in unprotected form) include but arenot limited to alkene, alkyne, alcohol, thiol, selenyl, phosphono,telluryl, cyano, amino, formyl, halo, boryl, and carboxylic acid surfaceattachment groups such as:

4-carboxyphenyl, carboxymethyl, 2-carboxyethyl, 3-carboxypropyl,2-(4-carboxyphenyl)ethynyl, 4-(2-(4-carboxyphenyl)ethynyl)phenyl,4-carboxymethylphenyl, 4-(3-carboxypropyl)phenyl,4-(2-(4-carboxymethylphenyl)ethynyl)phenyl; 4-hydroxyphenyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl,2-(4-hydroxyphenyl)ethynyl, 4-(2-(4-hydroxyphenyl)ethynyl)phenyl,4-hydroxymethylphenyl, 4-(2-hydroxyethyl)phenyl,4-(3-hydroxypropyl)phenyl, 4-(2-(4-hydroxymethylphenyl)ethynyl)phenyl;4-mercaptophenyl, mercaptomethyl, 2-mercaptoethyl, 3-mercaptopropyl,2-(4-mercaptophenyl)ethynyl, 4-(2-(4-mercaptophenyl)ethynyl)phenyl,4-mercaptomethylphenyl, 4-(2-mercaptoethyl)phenyl,4-(3-mercaptopropyl)phenyl, 4-(2-(4-mercaptomethylphenyl)ethynyl)phenyl;4-selenylphenyl, selenylmethyl, 2-selenylethyl, 3-selenylpropyl,2-(4-selenylphenyl)ethynyl, 4-selenylmethylphenyl,4-(2-selenylethyl)phenyl, 4-(3-selenylpropyl)phenyl,4-selenylmethylphenyl, 4-(2-(4-selenylphenyl)ethynyl)phenyl;4-tellurylphenyl, tellurylmethyl,2-tellurylethyl,3-tellurylpropyl,2-(4-tellurylphenyl)ethynyl, 4-(2-(4-tellurylphenyl)ethynyl)phenyl,4-tellurylmethylphenyl, 4-(2-tellurylethyl)phenyl,4-(3-tellurylpropyl)phenyl, 4-(2-(4-tellurylmethylphenyl)ethynyl)phenyl;

4-(dihydroxyphosphoryl)phenyl,(dihydroxyphosphoryl)methyl,2-(dihydroxyphosphoryl) ethyl,3-(dihydroxyphosphoryl)propyl, 2-[4-(dihydroxyphosphoryl)phenyl]ethynyl,4-[2-[4-(dihydroxyphosphoryephenyl]ethynyl]phenyl,4-[(dihydroxyphosphoryl)methyl]phenyl,4-[2-(dihydroxyphosphoryl)ethyl]phenyl,4-[2-[4-(dihydroxyphosphoryl)methylphenyl]ethynyl]phenyl;4-(hydroxy(mercapto)phosphoryl)phenyl,(hydroxy(mercapto)phosphoryl)methyl,2-(hydroxy(mercapto)phosphoryl)ethyl,3-(hydroxy(mercapto)phosphoryl)propyl,2-[4-(hydroxy(mercapto)phosphoryl)phenyl]ethynyl,4-[2-[4-(hydroxy(mercapto)phosphoryl)phenyl]ethynyl]phenyl,4-[(hydroxy(mercapto)phosphoryl)methyl]phenyl,4-[2-(hydroxy(mercapto)phosphoryl)ethyl]phenyl,4-[2-[4-(hydroxy(mercapto)phosphoryl)methylphenyl]ethynyl]phenyl;

4-cyanophenyl, cyanomethyl, 2-cyanoethyl, 3-cyanopropyl,2-(4-cyanophenyl)ethynyl, 4-[2-(4-cyanophenyl)ethynyl]phenyl,4-(cyanomethyl)phenyl, 4-(2-cyanoethyl)phenyl,4-[2-[4-(cyanomethyl)phenyl]ethynyl]phenyl;

4-cyanobiphenyl; 4-aminophenyl, aminomethyl, 2-aminoethyl,3-aminopropyl, 2-(4-aminophenyl)ethynyl,4-[2-(4-aminophenyl)ethynyl]phenyl, 4-aminobiphenyl;

4-formylphenyl, 4-bromophenyl, 4-iodophenyl, 4-vinylphenyl,4-ethynylphenyl, 4-allylphenyl, 4-[2-(trimethylsilyl)ethynyl]phenyl,4-[2-(triisopropylsilyl)ethynyl]phenyl,4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl;

formyl, bromo, iodo, bromomethyl, chloromethyl, ethynyl, vinyl, allyl;4-(ethynyl)biphen-4′-yl, 4-[2-(triisopropylsilyl)ethynyl]biphen-4′-yl,3,5-diethynylphenyl;

4-(bromomethyl)phenyl, and 2-bromoethyl.

In addition to the monodentate linker-surface attachment groupsdescribed above, multidentate linkers can be employed [Nikitin, K. Chem.Commun. 2003, 282-283; Hu, J.; Mattern, D. L. J. Org. Chem. 2000, 65,2277-2281; Yao, Y.; Tour, J. M. J. Org. Chem. 1999, 64, 1968-1971; Fox,M. A. et al. Langmuir, 1998, 14, 816-820; Galoppini, E.; Guo, W. J. Am.Chem. Soc. 2001, 123, 4342-4343; Deng, X. et al. J. Org. Chem. 2002, 67,5279-5283; Hector Jr., L. G. et al. Surface Science, 2001, 494, 1-20;Whitesell, J. K.; Chang, H. K. Science, 1993, 261, 73-76; Galoppini, E.et al. J. Am. Chem. Soc. 2002, 67, 7801-7811; Siiman, O. et al.Bioconjugate Chem. 2000, 11, 549-556]. Tripodal linkers bearing thiol,carboxylic acid, alcohol, or phosphoric acid units are particularlyattractive for firmly anchoring a molecular device on a planar surface.Specific examples of such linkers are built around the triphenylmethaneor tetraphenylmethane unit, including the following:

1,1,1-tris[4-(S-acetylthiomethyl)phenyl]methyl,

4-{1,1,1-tris[4-(S-acetylthiomethyl)phenyl]methyl}phenyl,

1,1,1-tris[4-(dihydroxyphosphoryl)phenyl]methyl,

4-{1,1,1-tris[4-(dihydroxyphosphoryl)phenyl]methyl}phenyl,

1,1,1-tris[4-dihydroxyphosphorylmethyl)phenyl]methyl, and

4-{1,1,1-tris[4-(dihydroxyphosphorylmethyl)phenyl]methyl}phenyl;

All as described in Balakumar, Muthukumaran and Lindsey, U.S. patentapplication Ser. No. 10/867,512 (filed Jun. 14, 2004). See also Lindsey,Loewe, Muthukumaran, and Ambroise, US Patent Application Publication No.20050096465 (Published May 5, 2005), particularly paragraph 51 thereof.Additional examples of multidentate linkers include but are not limitedto:

-   Alkene surface attachment groups (2, 3, 4 carbons) such as:

3-vinylpenta-1,4-dien-3-yl,

4-(3-vinylpenta-1,4-dien-3-yl)phenyl,

4-(3-vinylpenta-1,4-dien-3-yl)biphen-4′-yl,

4-allylhepta-1,6-dien-4-yl,

4-(4-allylhepta-1,6-dien-4-yl)phenyl,

4-(4-allylhepta-1,6-dien-4-yl)biphen-4′-yl,

5-(1-buten-4-yl)nona-1,8-dien-5-yl,

4-[5-(1-buten-4-yl)nona-1,8-dien-5-yl]phenyl,

4-[5-(1-buten-4-yl)nona-1,8-dien-5-yl]biphen-4′-yl, etc.

-   Alkyne surface attachment groups (2, 3, 4 carbons) such as:

3-ethynylpenta-1,4-diyn-3-yl,

4-(3-ethynylpenta-1,4-diyn-3-yl)phenyl,

4-(3-ethynylpenta-1,4-diyn-3-yl)biphen-4′-yl,

4-propargylhepta-1,6-diyn-4-yl,

4-(4-propargylhepta-1,6-diyn-4-yl)phenyl,

4-(4-propargylhepta-1,6-diyn-4-yl)biphen-4′-yl,

5-(1-butyn-4-yl)nona-1,8-diyn-5-yl,

4-[5-(1-butyn-4-yl)nona-1,8-diyn-5-yl]phenyl,

4-[5-(1-butyn-4-yl)nona-1,8-diyn-5-yl]biphen-4′-yl,

-   Alcohol surface attachment groups (1, 2, 3 carbons), such as:

2-(hydroxymethyl)-1,3-dihydroxyprop-2-yl,

4-[2-(hydroxymethyl)-1,3-dihydroxyprop-2-yl]phenyl,

4-[2-(hydroxymethyl)-1,3-dihydroxyprop-2-yl]biphen-4′-yl,

3-(2-hydroxyethyl)-1,5-dihydroxypent-3-yl,

4-[3-(2-hydroxyethyl)-1,5-dihydroxypent-3-yl]phenyl,

4-[3-(2-hydroxyethyl)-1,5-dihydroxypent-3-yl]biphen-4′-yl,

4-(3-hydroxypropyl)-1,7-dihydroxyhept-4-yl,

4-[4-(3-hydroxypropyl)-1,7-dihydroxyhept-4-yl]phenyl,

4-[4-(3-hydroxypropyl)-1,7-dihydroxyhept-4-yl]biphen-4′-yl, etc.,

-   Thiol surface attachment groups (1, 2, 3 carbons) such as:

2-(mercaptomethyl)-1,3-dimercaptoprop-2-yl,

4-[2-(mercaptomethyl)-1,3-dimercaptoprop-2-yl]phenyl,

4-[2-(mercaptomethyl)-1,3-dimercaptoprop-2-yl]biphen-4′-yl,

3-(2-mercaptoethyl)-1,5-dimercaptopent-3-yl

4-[3-(2-mercaptoethyl)-1,5-dimercaptopent-3-yl]phenyl,

4-[3-(2-mercaptoethyl)-1,5-dimercaptopent-3-yl]biphen-4′-yl,

4-(3-mercaptopropyl)-1,7-dimercaptohept-4-yl,

4-[4-(3-mercaptopropyl)-1,7-dimercaptohept-4-yl]phenyl,

4-[4-(3-mercaptopropyl)-1,7-dimercaptohept-4-yl]biphen-4′-yl etc.,

-   Selenyl surface attachment groups (1, 2, 3 carbons), such as:

2-(selenylmethyl)-1,3-diselenylprop-2-yl,

4-[2-(selenylmethyl)-1,3-diselenylprop-2-yl]phenyl,

4-[2-(mercaptomethyl)-1,3-dimercaptoprop-2-yl]biphen-4′-yl,

3-(2-selenylethyl)-1,5-diselenylpent-3-yl,

4-[3-(2-selenylethyl)-1,5-diselenylpent-3-yl]phenyl,

4-[3-(2-selenylethyl)-1,5-diselenylpent-3-yl]biphen-4′-yl,

4-(3-selenylpropyl)-1,7-diselenylhept-4-yl,

4-[4-(3-selenylpropyl)-1,7-diselenylhept-4-yl]phenyl,

4-[4-(3-selenylpropyl)-1,7-diselenylhept-4-yl]biphen-4′-yl, etc.

-   Phosphono surface attachment groups (1, 2, 3 carbons), such as:

2-(phosphonomethyl)-1,3-diphosphonoprop-2-yl,

4-[2-(phosphonomethyl)-1,3-diphosphonoprop-2-yl]phenyl,

4-[2-(phosphonomethyl)-1,3-diphosphonoprop-2-yl]biphen-4′-yl,

3-(2-phosphonoethyl)-1,5-diphosphonopent-3-yl,

4-[3-(2-phosphonoethyl)-1,5-diphosphonopent-3-yl]phenyl,

4-[3-(2-phosphonoethyl)-1,5-diphosphonopent-3-yl]biphen-4′-yl,

4-(3-phosphonopropyl)-1,7-diphosphonohept-4-yl,

4-[4-(3-phosphonopropyl)-1,7-diphosphonohept-4-yl]phenyl,

4-[4-(3-phosphonopropyl)-1,7-diphosphonohept-4-yl]biphen-4′-yl, etc.,and

-   Carboxylic acid surface attachment groups (1, 2, 3 carbons), such    as:

2-(carboxymethyl)-1,3-dicarboxyprop-2-yl,

4-[2-(carboxymethyl)-1,3-dicarboxyprop-2-yl]phenyl,

4-[2-(carboxymethyl)-1,3-dicarboxyprop-2-yl]biphen-4′-yl,

3-(2-carboxyethyl)-1,5-dicarboxypent-3-yl,

4-[3-(2-carboxyethyl)-1,5-dicarboxypent-3-yl]phenyl,

4-[3-(2-carboxyethyl)-1,5-dicarboxypent-3-yl]biphen-4′-yl,

4-(3-carboxypropyl)-1,7-dicarboxyhept-4-yl,

4-[4-(3-carboxypropyl)-1,7-dicarboxyhept-4-yl]phenyl,

4-[4-(3-carboxypropyl)-1,7-dicarboxyhept-4-yl]biphen-4′-yl, etc.

It is to be understood that the compounds provided herein may containchiral centers. Such chiral centers may be of either the (R) or (S)configuration, or may be a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, or be stereoisomeric ordiastereomeric mixtures. It is to be understood that the chiral centersof the compounds provided herein may undergo epimerization in vivo. Assuch, one of skill in the art will recognize that administration of acompound in its (R) form is equivalent, for compounds that undergoepimerization in vivo, to administration of the compound in its (S)form.

Active compounds of the invention can be provided as pharmaceuticallyacceptable salts. Such salts include, but are not limited to, aminesalts, such as but not limited to N,N′-dibenzylethylenediamine,chloroprocaine, choline, ammonia, diethanolamine and otherhydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine,N-benzylphenethylamine,1-para-chlorobenzyl-2-pyrrolidin-1′-ylmethyl-benzimidazole, diethylamineand other alkylamines, piperazine and tris(hydroxymethyl)aminomethane;alkali metal salts, such as but not limited to lithium, potassium andsodium; alkali earth metal salts, such as but not limited to barium,calcium and magnesium; transition metal salts, such as but not limitedto zinc; and other metal salts, such as but not limited to sodiumhydrogen phosphate and disodium phosphate; and also including, but notlimited to, salts of mineral acids, such as but not limited tohydrochlorides and sulfates; and salts of organic acids, such as but notlimited to acetates, lactates, malates, tartrates, citrates, ascorbates,succinates, butyrates, valerates and fumarates. Pharmaceuticallyacceptable esters include, but are not limited to, alkyl, alkenyl,alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl andheterocyclyl esters of acidic groups, including, but not limited to,carboxylic acids, phosphoric acids, phosphinic acids, sulfonic acids,sulfinic acids and boronic acids.

Active compounds of the invention include prodrugs of the compoundsdescribed herein. As noted above, a “prodrug” is a compound that, uponin vivo administration, is metabolized by one or more steps or processesor otherwise converted to the biologically, pharmaceutically ortherapeutically active form of the compound. To produce a prodrug, thepharmaceutically active compound is modified such that the activecompound will be regenerated by metabolic processes. The prodrug may bedesigned to alter the metabolic stability or the transportcharacteristics of a drug, to mask side effects or toxicity, to improvethe flavor of a drug or to alter other characteristics or properties ofa drug. By virtue of knowledge of pharmacodynamic processes and drugmetabolism in vivo, those of skill in this art, once a pharmaceuticallyactive compound is known, can design prodrugs of the compound (see,e.g., Nogrady (1985) Medicinal Chemistry A Biochemical Approach, OxfordUniversity Press, New York, pages 388-392).

Utility. The methods and intermediates described herein are useful forthe synthesis of compounds of Formula I as described herein. Suchcompounds are useful per se or in further modified form (e.g., as asalt, metalated compound, conjugate or prodrug) for diagnostic andtherapeutic purposes in like manner as other compounds described forphotodynamic therapy, such as described in US Patent ApplicationPublication No. 2004/0044197 to Pandey et al. and as set forth infurther detail below.

Stability. An advantage of the compounds of the present invention istheir stability and absorption characteristics. Thus, the presentinvention provides a “neat” composition consisting of an active compoundof the invention (e.g., compounds of Formula I, or the pharmaceuticallyacceptable salts, prodrugs, or conjugates thereof (e.g., with atargeting agent such as a protein, peptide or antibody)), wherein thecomposition has or is characterized by a peak Molar absorptioncoefficient in solution of at least 10,000, up to 300,000 M⁻¹cm⁻¹ ormore, at a wavelength between 650 and 850 or 900 nanometers (it beingunderstood that (a) the active compound must be placed into solution todetermine its peak Molar absorption coefficient at the indicatedwavelength; and (b) the compound may exhibit additional peaks outside ofthis range, or multiple peaks within this range).

In addition, the present invention provides compositions comprising orconsisting essentially of an active compound of the invention (e.g.,compounds of Formula I, or the pharmaceutically acceptable salts,prodrugs, or conjugates thereof (e.g., with a targeting agent such as aprotein, peptide or antibody)) in a solvent. The amount of solvent isnot critical and may comprise from 0.01 or 1 to 99 or 99.99 percent byweight of the composition. The composition has or is characterized by apeak Molar absorption coefficient in solution of at least 10,000, up to300,000 M⁻¹cm⁻¹ or more, at a wavelength between 650 and 850 or 900nanometers. It will be appreciated that agitation may be required tobreak agglomerated particles back into solution prior to determiningmolar absorption, but that some level of agglomeration may actually bedesired for practical use of the composition. Suitable solvents dependupon the particular compound and intended use for that compound, butinclude both organic solvents, aqueous solvents and combinationsthereof.

The compositions, be they the bacteriochlorin compounds in “neat” formor the compounds mixed with a solvent, have or exhibit a loss of notmore than 10, 15 or 20 percent by weight of the bacteriochlorin compoundof the invention (due to degredation thereof) when stored in a sealedvessel (e.g., a flask ampoule or vial), at room temperature in theabsence of ambient light for at least 3 or 4 months. Degredation can bedetermined by spectroscopy, thin-layer chromatography, NMR spectroscopy,and/or mass spectrometry, in accordance with known techniques.

2. Pharmaceutical Formulations.

Formulation of Pharmaceutical Compositions. The pharmaceuticalcompositions provided herein contain therapeutically effective amountsof one or more of the compounds provided herein that are useful in theprevention, treatment, or amelioration of one or more of the symptoms ofdiseases or disorders associated with hyperproliferating tissue orneovascularization, or in which hyperproliferating tissue orneovascularization is implicated, in a pharmaceutically acceptablecarrier. Diseases or disorders associated with hyperproliferating tissueor neovascularization include, but are not limited to, cancer,psoriasis, atherosclerosis, heart disease, and age-related maculardegeneration. Pharmaceutical carriers suitable for administration of thecompounds provided herein include any such carriers known to thoseskilled in the art to be suitable for the particular mode ofadministration.

Pharmaceutical compositions preferably exhibit the absorptioncharacteristics and storage or stability characteristics describedabove.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients.

The compositions contain one or more compounds provided herein. Thecompounds are, in one embodiment, formulated into suitablepharmaceutical preparations such as solutions, suspensions, tablets,dispersible tablets, pills, capsules, powders, sustained releaseformulations or elixirs, for oral administration or in sterile solutionsor suspensions for parenteral administration, as well as transdermalpatch preparation and dry powder inhalers. In one embodiment, thecompounds described above are formulated into pharmaceuticalcompositions using techniques and procedures well known in the art (see,e.g., Ansel, Introduction to Pharmaceutical Dosage Forms, Fourth Edition1985, 126).

In the compositions, effective concentrations of one or more compoundsor pharmaceutically acceptable derivatives thereof is (are) mixed with asuitable pharmaceutical carrier. The compounds may be derivatized as thecorresponding salts, esters, enol ethers or esters, acetals, ketals,orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydratesor prodrugs prior to formulation, as described above. The concentrationsof the compounds in the compositions are effective for delivery of anamount, upon administration, that treats, prevents, or ameliorates oneor more of the symptoms of diseases or disorders associated withhyperproliferating tissue or neovascularization or in whichhyperproliferating tissue or neovascularization is implicated.

In one embodiment, the compositions are formulated for single dosageadministration. To formulate a composition, the weight fraction ofcompound is dissolved, suspended, dispersed or otherwise mixed in aselected carrier at an effective concentration such that the treatedcondition is relieved, prevented, or one or more symptoms areameliorated.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in in vitro and in vivo systems described hereinand in U.S. Pat. No. 5,952,366 to Pandey et al. (1999) and thenextrapolated therefrom for dosages for humans.

The concentration of active compound in the pharmaceutical compositionwill depend on absorption, inactivation and excretion rates of theactive compound, the physicochemical characteristics of the compound,the dosage schedule, and amount administered as well as other factorsknown to those of skill in the art. For example, the amount that isdelivered is sufficient to ameliorate one or more of the symptoms ofdiseases or disorders associated with hyperproliferating tissue orneovascularization or in which hyperproliferating tissue orneovascularization is implicated, as described herein.

In one embodiment, a therapeutically effective dosage should produce aserum concentration of active ingredient of from about 0.1 ng/ml toabout 50-100 ug/ml. In one embodiment, a therapeutically effectivedosage is from 0.001, 0.01 or 0.1 to 10, 100 or 1000 mg of activecompound per kilogram of body weight per day. Pharmaceutical dosage unitforms are prepared to provide from about 0.01 mg, 0.1 mg or 1 mg toabout 500 mg, 1000 mg or 2000 mg, and in one embodiment from about 10 mgto about 500 mg of the active ingredient or a combination of essentialingredients per dosage unit form.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed compositions.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as TWEEN™, or dissolution in aqueous sodium bicarbonate.Derivatives of the compounds, such as prodrugs of the compounds may alsobe used in formulating effective pharmaceutical compositions.

Upon mixing or addition of the compound(s), the resulting mixture may bea solution, suspension, emulsion or the like. The form of the resultingmixture depends upon a number of factors, including the intended mode ofadministration and the solubility of the compound in the selectedcarrier or vehicle. The effective concentration is sufficient forameliorating the symptoms of the disease, disorder or condition treatedand may be empirically determined.

The pharmaceutical compositions are provided for administration tohumans and animals in unit dosage forms, such as tablets, capsules,pills, powders, granules, sterile parenteral solutions or suspensions,and oral solutions or suspensions, and oil-water emulsions containingsuitable quantities of the compounds or pharmaceutically acceptablederivatives thereof. The pharmaceutically therapeutically activecompounds and derivatives thereof are, in one embodiment, formulated andadministered in unit-dosage forms or multiple-dosage forms. Unit-doseforms as used herein refers to physically discrete units suitable forhuman and animal subjects and packaged individually as is known in theart. Each unit-dose contains a predetermined quantity of thetherapeutically active compound sufficient to produce the desiredtherapeutic effect, in association with the required pharmaceuticalcarrier, vehicle or diluent. Examples of unit-dose forms includeampoules and syringes and individually packaged tablets or capsules.Unit-dose forms may be administered in fractions or multiples thereof. Amultiple-dose form is a plurality of identical unit-dosage formspackaged in a single container to be administered in segregatedunit-dose form. Examples of multiple-dose forms include vials, bottlesof tablets or capsules or bottles of pints or gallons. Hence, multipledose form is a multiple of unit-doses which are not segregated inpackaging.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, or otherwise mixing an activecompound as defined above and optional pharmaceutical adjuvants in acarrier, such as, for example, water, saline, aqueous dextrose,glycerol, glycols, ethanol, and the like, to thereby form a solution orsuspension. If desired, the pharmaceutical composition to beadministered may also contain minor amounts of nontoxic auxiliarysubstances such as wetting agents, emulsifying agents, solubilizingagents, pH buffering agents and the like, for example, acetate, sodiumcitrate, cyclodextrine derivatives, sorbitan monolaurate,triethanolamine sodium acetate, triethanolamine oleate, and other suchagents.

Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in this art; for example, see Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 15thEdition, 1975.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. Methods for preparation of these compositions are known tothose skilled in the art. The contemplated compositions may contain0.001%-100% active ingredient, in one embodiment 0.1-95%, in anotherembodiment 75-85%.

Compositions for Oral Administration. Oral pharmaceutical dosage formsare either solid, gel or liquid. The solid dosage forms are tablets,capsules, granules, and bulk powders. Types of oral tablets includecompressed, chewable lozenges and tablets which may be enteric-coated,sugar-coated or film-coated. Capsules may be hard or soft gelatincapsules, while granules and powders may be provided in non-effervescentor effervescent form with the combination of other ingredients known tothose skilled in the art.

Solid Compositions for Oral Administration. In certain embodiments, theformulations are solid dosage forms, in one embodiment, capsules ortablets. The tablets, pills, capsules, troches and the like can containone or more of the following ingredients, or compounds of a similarnature: a binder; a lubricant; a diluent; a glidant; a disintegratingagent; a coloring agent; a sweetening agent; a flavoring agent; awetting agent; an emetic coating; and a film coating. Examples ofbinders include microcrystalline cellulose, gum tragacanth, glucosesolution, acacia mucilage, gelatin solution, molasses,polvinylpyrrolidine, povidone, crospovidones, sucrose and starch paste.Lubricants include talc, starch, magnesium or calcium stearate,lycopodium and stearic acid. Diluents include, for example, lactose,sucrose, starch, kaolin, salt, mannitol and dicalcium phosphate.Glidants include, but are not limited to, colloidal silicon dioxide.Disintegrating agents include crosscarmellose sodium, sodium starchglycolate, alginic acid, corn starch, potato starch, bentonite,methylcellulose, agar and carboxymethylcellulose. Coloring agentsinclude, for example, any of the approved certified water soluble FD andC dyes, mixtures thereof; and water insoluble FD and C dyes suspended onalumina hydrate. Sweetening agents include sucrose, lactose, mannitoland artificial sweetening agents such as saccharin, and any number ofspray dried flavors. Flavoring agents include natural flavors extractedfrom plants such as fruits and synthetic blends of compounds whichproduce a pleasant sensation, such as, but not limited to peppermint andmethyl salicylate. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelaural ether. Emetic-coatings include fatty acids, fats, waxes, shellac,ammoniated shellac and cellulose acetate phthalates. Film coatingsinclude hydroxyethylcellulose, gellan gum, sodiumcarboxymethylcellulose, polyethylene glycol 4000 and cellulose acetatephthalate.

The compound, or pharmaceutically acceptable derivative thereof, couldbe provided in a composition that protects it from the acidicenvironment of the stomach. For example, the composition can beformulated in an enteric coating that maintains its integrity in thestomach and releases the active compound in the intestine. Thecomposition may also be formulated in combination with an antacid orother such ingredient. When the dosage unit form is a capsule, it cancontain, in addition to material of the above type, a liquid carriersuch as a fatty oil. In addition, dosage unit forms can contain variousother materials which modify the physical form of the dosage unit, forexample, coatings of sugar and other enteric agents. The compounds canalso be administered as a component of an elixir, suspension, syrup,wafer, sprinkle, chewing gum or the like. A syrup may contain, inaddition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The active materials can also be mixed with other active materials whichdo not impair the desired action, or with materials that supplement thedesired action, such as antacids, H2 blockers, and diuretics. The activeingredient is a compound or pharmaceutically acceptable derivativethereof as described herein. Higher concentrations, up to about 98% byweight of the active ingredient may be included.

In all embodiments, tablets and capsules formulations may be coated asknown by those of skill in the art in order to modify or sustaindissolution of the active ingredient. Thus, for example, they may becoated with a conventional enterically digestible coating, such asphenylsalicylate, waxes and cellulose acetate phthalate.

Liquid Compositions for Oral Administration. Liquid oral dosage formsinclude aqueous solutions, emulsions, suspensions, solutions and/orsuspensions reconstituted from non-effervescent granules andeffervescent preparations reconstituted from effervescent granules.Aqueous solutions include, for example, elixirs and syrups. Emulsionsare either oil-in-water or water-in-oil.

Elixirs are clear, sweetened, hydroalcoholic preparations.Pharmaceutically acceptable carriers used in elixirs include solvents.Syrups are concentrated aqueous solutions of a sugar, for example,sucrose, and may contain a preservative. An emulsion is a two-phasesystem in which one liquid is dispersed in the form of small globulesthroughout another liquid. Pharmaceutically acceptable carriers used inemulsions are non-aqueous liquids, emulsifying agents and preservatives.Suspensions use pharmaceutically acceptable suspending agents andpreservatives. Pharmaceutically acceptable substances used innon-effervescent granules, to be reconstituted into a liquid oral dosageform, include diluents, sweeteners and wefting agents. Pharmaceuticallyacceptable substances used in effervescent granules, to be reconstitutedinto a liquid oral dosage form, include organic acids and a source ofcarbon dioxide. Coloring and flavoring agents are used in all of theabove dosage forms. Solvents include glycerin, sorbitol, ethyl alcoholand syrup. Examples of preservatives include glycerin, methyl andpropylparaben, benzoic acid, sodium benzoate and alcohol. Examples ofnon-aqueous liquids utilized in emulsions include mineral oil andcottonseed oil. Examples of emulsifying agents include gelatin, acacia,tragacanth, bentonite, and surfactants such as polyoxyethylene sorbitanmonooleate. Suspending agents include sodium carboxymethylcellulose,pectin, tragacanth, xanthan gum, Veegum and acacia. Sweetening agentsinclude sucrose, syrups, glycerin and artificial sweetening agents suchas saccharin. Wetting agents include propylene glycol monostearate,sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylenelauryl ether. Organic acids include citric and tartaric acid. Sources ofcarbon dioxide include sodium bicarbonate and sodium carbonate. Coloringagents include any of the approved certified water soluble FD and Cdyes, and mixtures thereof. Flavoring agents include natural flavorsextracted from plants such fruits, and synthetic blends of compoundswhich produce a pleasant taste sensation. For a solid dosage form, thesolution or suspension, in for example propylene carbonate, vegetableoils or triglycerides, is in one embodiment encapsulated in a gelatincapsule. Such solutions, and the preparation and encapsulation thereof,are disclosed in U.S. Pat. Nos. 4,328,245; 4,409,239; and 4,410,545. Fora liquid dosage form, the solution, e.g., for example, in a polyethyleneglycol, may be diluted with a sufficient quantity of a pharmaceuticallyacceptable liquid carrier, e.g., water, to be easily measured foradministration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and other such carriers, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells. Other usefulformulations include those set forth in U.S. Pat. Nos. RE28,819 and4,358,603. Briefly, such formulations include, but are not limited to,those containing a compound provided herein, a dialkylated mono- orpoly-alkylene glycol, including, but not limited to,1,2-dimethoxymethane, diglyme, triglyme, tetraglyme, polyethyleneglycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether,polyethylene glycol-750-dimethyl ether wherein 350, 550 and 750 refer tothe approximate average molecular weight of the polyethylene glycol, andone or more antioxidants, such as butylated hydroxytoluene (BHT),butylated hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone,hydroxycoumarins, ethanolamine, lecithin, cephalin, ascorbic acid, malicacid, sorbitol, phosphoric acid, thiodipropionic acid and its esters,and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholicsolutions including a pharmaceutically acceptable acetal. Alcohols usedin these formulations are any pharmaceutically acceptable water-misciblesolvents having one or more hydroxyl groups, including, but not limitedto, propylene glycol and ethanol. Acetals include, but are not limitedto, di(lower alkyl) acetals of lower alkyl aldehydes such asacetaldehyde diethyl acetal.

3. Injectables, Solutions and Emulsions. Parenteral administration, inone embodiment characterized by injection, either subcutaneously,intramuscularly or intravenously is also contemplated herein.Injectables can be prepared in conventional forms, either as liquidsolutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Theinjectables, solutions and emulsions also contain one or moreexcipients. Suitable excipients are, for example, water, saline,dextrose, glycerol or ethanol. In addition, if desired, thepharmaceutical compositions to be administered may also contain minoramounts of non-toxic auxiliary substances such as wetting or emulsifyingagents, pH buffering agents, stabilizers, solubility enhancers, andother such agents, such as for example, sodium acetate, sorbitanmonolaurate, triethanolamine oleate and cyclodextrins.

Implantation of a slow-release or sustained-release system, such that aconstant level of dosage is maintained (see, e.g., U.S. Pat. No.3,710,795) is also contemplated herein. Briefly, a compound providedherein is dispersed in a solid inner matrix, e.g.,polymethylmethacrylate, polybutylmethacrylate, plasticized orunplasticized polyvinylchloride, plasticized nylon, plasticizedpolyethyleneterephthalate, natural rubber, polyisoprene,polyisobutylene, polybutadiene, polyethylene, ethylene-vinylacetatecopolymers, silicone rubbers, polydimethylsiloxanes, silicone carbonatecopolymers, hydrophilic polymers such as hydrogels of esters of acrylicand methacrylic acid, collagen, cross-linked polyvinylalcohol andcross-linked partially hydrolyzed polyvinyl acetate, that is surroundedby an outer polymeric membrane, e.g., polyethylene, polypropylene,ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers,ethylene/vinylacetate copolymers, silicone rubbers, polydimethylsiloxanes, neoprene rubber, chlorinated polyethylene, polyvinylchloride,vinylchloride copolymers with vinyl acetate, vinylidene chloride,ethylene and propylene, ionomer polyethylene terephthalate, butyl rubberepichlorohydrin rubbers, ethylene/vinyl alcohol copolymer,ethylene/vinyl acetate/vinyl alcohol terpolymer, andethylene/vinyloxyethanol copolymer, that is insoluble in body fluids.The compound diffuses through the outer polymeric membrane in a releaserate controlling step. The percentage of active compound contained insuch parenteral compositions is highly dependent on the specific naturethereof, as well as the activity of the compound and the needs of thesubject.

Parenteral administration of the compositions includes intravenous,subcutaneous and intramuscular administrations. Preparations forparenteral administration include sterile solutions ready for injection,sterile dry soluble products, such as lyophilized powders, ready to becombined with a solvent just prior to use, including hypodermic tablets,sterile suspensions ready for injection, sterile dry insoluble productsready to be combined with a vehicle just prior to use and sterileemulsions. The solutions may be either aqueous or nonaqueous.

If administered intravenously, suitable carriers include physiologicalsaline or phosphate buffered saline (PBS), and solutions containingthickening and solubilizing agents, such as glucose, polyethyleneglycol, and polypropylene glycol and mixtures thereof.

Pharmaceutically acceptable carriers used in parenteral preparationsinclude aqueous vehicles, nonaqueous vehicles, antimicrobial agents,isotonic agents, buffers, antioxidants, local anesthetics, suspendingand dispersing agents, emulsifying agents, sequestering or chelatingagents and other pharmaceutically acceptable substances.

Examples of aqueous vehicles include Sodium Chloride Injection, RingersInjection, Isotonic Dextrose Injection, Sterile Water Injection,Dextrose and Lactated Ringers Injection. Nonaqueous parenteral vehiclesinclude fixed oils of vegetable origin, cottonseed oil, corn oil, sesameoil and peanut oil. Antimicrobial agents in bacteriostatic orfungistatic concentrations must be added to parenteral preparationspackaged in multiple-dose containers which include phenols or cresols,mercurials, benzyl alcohol, chlorobutanol, methyl and propylp-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride andbenzethonium chloride. Isotonic agents include sodium chloride anddextrose. Buffers include phosphate and citrate. Antioxidants includesodium bisulfate. Local anesthetics include procaine hydrochloride.Suspending and dispersing agents include sodium carboxymethylcelluose,xanthan gum, hydroxypropyl methylcellulose and polyvinylpyrrolidone.Emulsifying agents include Polysorbate 80 (TWEEN™ 80). A sequestering orchelating agent of metal ions includes EDTA. Pharmaceutical carriersalso include ethyl alcohol, polyethylene glycol and propylene glycol forwater miscible vehicles; and sodium hydroxide, hydrochloric acid, citricacid or lactic acid for pH adjustment.

The concentration of the pharmaceutically active compound is adjusted sothat an injection provides an effective amount to produce the desiredpharmacological effect. The exact dose depends on the age, weight andcondition of the patient or animal as is known in the art.

The unit-dose parenteral preparations are packaged in an ampoule, a vialor a syringe with a needle. All preparations for parenteraladministration must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intraarterial infusion of a sterileaqueous solution containing an active compound is an effective mode ofadministration. Another embodiment is a sterile aqueous or oily solutionor suspension containing an active material injected as necessary toproduce the desired pharmacological effect.

Injectables are designed for local and systemic administration. In oneembodiment, a therapeutically effective dosage is formulated to containa concentration of at least about 0.1% w/w up to about 90% w/w or more,in certain embodiments more than 1% w/w of the active compound to thetreated tissue(s).

The compound may be suspended in micronized or other suitable form ormay be derivatized to produce a more soluble active product or toproduce a prodrug. The form of the resulting mixture depends upon anumber of factors, including the intended mode of administration and thesolubility of the compound in the selected carrier or vehicle. Theeffective concentration is sufficient for ameliorating the symptoms ofthe condition and may be empirically determined.

Lyophilized Powders. Lyophilized powders, which can be reconstituted foradministration as solutions, emulsions and other mixtures, can also beused to carry out the present invention. They may also be reconstitutedand formulated as solids or gels.

The sterile, lyophilized powder is prepared by dissolving a compoundprovided herein, or a pharmaceutically acceptable derivative thereof, ina suitable solvent. The solvent may contain an excipient which improvesthe stability or other pharmacological component of the powder orreconstituted solution, prepared from the powder. Excipients that may beused include, but are not limited to, dextrose, sorbital, fructose, cornsyrup, xylitol, glycerin, glucose, sucrose or other suitable agent. Thesolvent may also contain a buffer, such as citrate, sodium or potassiumphosphate or other such buffer known to those of skill in the art at, inone embodiment, about neutral pH. Subsequent sterile filtration of thesolution followed by lyophilization under standard conditions known tothose of skill in the art provides the desired formulation. In oneembodiment, the resulting solution will be apportioned into vials forlyophilization. Each vial will contain a single dosage or multipledosages of the compound. The lyophilized powder can be stored underappropriate conditions, such as at about 4° C. to room temperature.

Reconstitution of this lyophilized powder with water for injectionprovides a formulation for use in parenteral administration. Forreconstitution, the lyophilized powder is added to sterile water orother suitable carrier. The precise amount depends upon the selectedcompound. Such amount can be empirically determined.

Topical Administration. Topical mixtures are prepared as described forthe local and systemic administration. The resulting mixture may be asolution, suspension, emulsions or the like and are formulated ascreams, gels, ointments, emulsions, solutions, elixirs, lotions,suspensions, tinctures, pastes, foams, aerosols, irrigations, sprays,suppositories, bandages, dermal patches or any other formulationssuitable for topical administration.

The compounds or pharmaceutically acceptable derivatives thereof may beformulated as aerosols for topical application, such as by inhalation(see, e.g., U.S. Pat. Nos. 4,044,126; 4,414,209; and 4,364,923, whichdescribe aerosols for delivery of a steroid useful for treatment ofinflammatory diseases, particularly asthma). These formulations foradministration to the respiratory tract can be in the form of an aerosolor solution for a nebulizer, or as a microfine powder for insufflation,alone or in combination with an inert carrier such as lactose. In such acase, the particles of the formulation will, in one embodiment, havediameters of less than 50 microns, in one embodiment less than 10microns.

The compounds may be formulated for local or topical application, suchas for topical application to the skin and mucous membranes, such as inthe eye, in the form of gels, creams, and lotions and for application tothe eye or for intracisternal or intraspinal application. Topicaladministration is contemplated for transdermal delivery and also foradministration to the eyes or mucosa, or for inhalation therapies. Nasalsolutions of the active compound alone or in combination with otherpharmaceutically acceptable excipients can also be administered. Thesesolutions, particularly those intended for ophthalmic use, may beformulated as 0.01% -10% isotonic solutions, pH about 5-7, withappropriate salts.

Compositions for other Routes of Administration. Other routes ofadministration, such as transdermal patches, including iontophoretic andelectrophoretic devices, and rectal administration, are alsocontemplated herein.

Transdermal patches, including iotophoretic and electrophoretic devices,are well known to those of skill in the art. For example, such patchesare disclosed in U.S. Pat. Nos. 6,267,983; 6,261,595; 6,256,533;6,167,301; 6,024,975; 6,010715; 5,985,317; 5,983,134; 5,948,433 and5,860,957.

For example, pharmaceutical dosage forms for rectal administration arerectal suppositories, capsules and tablets for systemic effect. Rectalsuppositories are used herein mean solid bodies for insertion into therectum which melt or soften at body temperature releasing one or morepharmacologically or therapeutically active ingredients.Pharmaceutically acceptable substances utilized in rectal suppositoriesare bases or vehicles and agents to raise the melting point. Examples ofbases include cocoa butter (theobroma oil), glycerin-gelatin, carbowax(polyoxyethylene glycol) and appropriate mixtures of mono-, di- andtriglycerides of fatty acids. Combinations of the various bases may beused. Agents to raise the melting point of suppositories includespermaceti and wax. Rectal suppositories may be prepared either by thecompressed method or by molding. The weight of a rectal suppository, inone embodiment, is about 2 to 3 gm.

Tablets and capsules for rectal administration are manufactured usingthe same pharmaceutically acceptable substance and by the same methodsas for formulations for oral administration.

Targeted Formulations. The compounds provided herein, orpharmaceutically acceptable derivatives thereof, may also be formulatedto be targeted to a particular tissue, receptor, infecting agent orother area of the body of the subject to be treated. Many such targetingmethods are well known to those of skill in the art. All such targetingmethods are contemplated herein for use in the instant compositions. Fornon-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos.6,316,652; 6,274,552; 6,271,359; 6,253,872; 6,139,865; 6,131,570;6,120,751; 6,071,495; 6,060,082; 6,048,736; 6,039,975; 6,004,534;5,985,307; 5,972,366; 5,900,252; 5,840,674; 5,759,542 and 5,709,874.

Liposomes. In one embodiment, liposomal suspensions, includingtissue-targeted liposomes, such as tumor-targeted liposomes, may also besuitable as pharmaceutically acceptable carriers. These may be preparedaccording to methods known to those skilled in the art. For example,liposome formulations may be prepared as described in U.S. Pat. No.4,522,811. Briefly, liposomes such as multilamellar vesicles (MLV's) maybe formed by drying down egg phosphatidyl choline and brain phosphatidylserine (7:3 molar ratio) on the inside of a flask. A solution of acompound provided herein in phosphate buffered saline lacking divalentcations (PBS) is added and the flask shaken until the lipid film isdispersed. The resulting vesicles are washed to remove unencapsulatedcompound, pelleted by centrifugation, and then resuspended in PBS.

Ligands. In another embodiment, the disclosed compounds may be targetedto specific target tissues or target compositions using ligands specificfor the target tissue or target composition, for example, using ligandsor ligand-receptor pairs such as antibodies and antigens. Antibodiesagainst tumor antigens and against pathogens are known. For example,antibodies and antibody fragments which specifically bind markersproduced by or associated with tumors or infectious lesions, includingviral, bacterial, fungal and parasitic infections, and antigens andproducts associated with such microorganisms have been disclosed, interalia, in Hansen et al., U.S. Pat. No. 3,927,193 and Goldenberg, U.S.Pat. Nos. 4,331,647; 4,348,376; 4,361,544; 4,468,457; 4,444,744;4,818,709 and 4,624,846. Antibodies against an antigen, e.g., agastrointestinal, lung, breast, prostate, ovarian, testicular, brain orlymphatic tumor, a sarcoma or a melanoma, can be used.

A wide variety of monoclonal antibodies against infectious diseaseagents have been developed, and are summarized in a review by Polin, inEur. J. Clin. Microbiol., 3(5): 387-398 (1984), showing readyavailability. These include monoclonal antibodies (MAbs) againstpathogens and their antigens such as the following: Anti-bacterial Mabssuch as those against Streptococcus agalactiae, Legionella pneumophilia,Streptococcus pyogenes, Esherichia coli, Neisseria gonorrhosae,Neisseria meningitidis, Pneumococcus, Hemophilis influenzae B, Treponemapallidum, Lyme disease, spirochetes, Pseudomonas aeruginosa,Mycobacterium leprae, Brucella abortus, Mycobacterium tuberculosis,Tetanus toxin, Anti-protozoan Mabs such as those against Plasmodiumfalciparum, Plasmodium vivax, Toxoplasma gondii, Trypanosoma rangeli,Trypanosoma cruzi, Trypanosoma rhodesiensei, Trypanosoma brucei,Schistosoma mansoni, Schistosoma japanicum, Mesocestoides corti, Emeriatenella, Onchocerca volvulus, Leishmania tropica, Trichinella spiralis,Theileria parva, Taenia hydatigena, Taenia ovis, Taenia saginata,Anti-viral MAbs such as those against HIV-1, -2, and -3, Hepatitis A, B,C, D, Rabies virus, Influenza virus, Cytomegalovirus, Herpes simplex Iand II, Human serum parvo-like virus, Respiratory syncytial virus,Varicella-Zoster virus, Hepatitis B virus, Measles virus, Adenovirus,Human T-cell leukemia viruses, Epstein-Barr virus, Mumps virus, Sindbisvirus, Mouse mammary tumor virus, Feline leukemia virus, Lymphocyticchoriomeningitis virus, Wart virus, Blue tongue virus, Sendai virus, Reovirus, Polio virus, Dengue virus, Rubella virus, Murine leukemia virus,Antimycoplasmal MAbs such as those against Acholeplasma laidlawii,Mycoplasma arthritidis, M. hyorhinis, M. orale, M. arginini, M.pneumonia; etc.

Suitable MAbs have been developed against most of the micro-organisms(bacteria, viruses, protozoa, other parasites) responsible for themajority of infections in humans, and many have been used previously forin vitro diagnostic purposes. These antibodies, and newer MAbs that canbe generated by conventional methods, are appropriate for use as targetagents with the compounds provided herein.

MAbs against malaria parasites can be directed against the sporozoite,merozoite, schizont and gametocyte stages. Monoclonal antibodies havebeen generated against sporozoites (circumsporozoite antigen), and havebeen shown to neutralize sporozoites in vitro and in rodents (N. Yoshidaet al., Science 207: 71-73 (1980)). Monoclonal antibodies to T. gondii,the protozoan parasite involved in toxoplasmosis have been developed(Kasper et al., J. Immunol. 129: 1694-1699 (1982). MAbs have beendeveloped against schistosomular surface antigens and have been found toact against schistosomulae in vivo or in vitro (Simpson et al.,Parasitology 83: 163-177 (1981); Smith et al., Parasitology 84: 83-91(1982); Gryzch et al., J. Immunol. 129: 2739-2743 (1982); Zodda et al.,J. Immunol. 129: 2326-2328 (1982); Dissous et al., J. Immunol. 129:2232-2234 (1982).

It should be noted that mixtures of antibodies andimmunoglobulin-classes can be used, as can hybrid antibodies.Multispecific, including bispecific and hybrid, antibodies and antibodyfragments are especially preferred in the methods of the presentinvention for detecting and treating target tissue and are comprised ofat least two different substantially monospecific antibodies or antibodyfragments, wherein at least two of said antibodies or antibody fragmentsspecifically bind to at least two different antigens produced orassociated with the targeted lesion or at least two different epitopesor molecules of a marker substance produced or associated with thetarget tissue. Multispecific antibodies and antibody fragments with dualspecificities can be prepared analogously to the anti-tumor markerhybrids disclosed in U.S. Pat. No. 4,361,544. Other techniques forpreparing hybrid antibodies are disclosed in, e.g., U.S. Pat. Nos.4,474,893 and 4,479,895, and in Milstein et al., Immunol. Today 5: 299(1984).

Antibody fragments useful in the present invention include F(ab′)₂,F(ab)₂, Fab′, Fab, Fv and the like including hybrid fragments. Preferredfragments are Fab′, F(ab′)₂, Fab, and F(ab)₂. Also useful are anysubfragments retaining the hypervariable, antigen-binding region of animmunoglobulin and having a size similar to or smaller than a Fab′fragment. This will include genetically engineered and/or recombinantproteins, whether single-chain or multiple-chain, which incorporate anantigen-binding site and otherwise function in vivo as targetingvehicles in substantially the same way as natural immunoglobulinfragments. Such single-chain binding molecules are disclosed in U.S.Pat. No. 4,946,778, which is hereby incorporated by reference. Fab′antibody fragments may be conveniently made by reductive cleavage ofF(ab′)₂ fragments, which themselves may be made by pepsin digestion ofintact immunoglobulin Fab antibody fragments may be made by papaindigestion of intact immunoglobulin, under reducing conditions, or bycleavage of F(ab)₂ fragments which result from careful papain digestionof whole immunoglobulin.

A ligand or one member of a ligand-receptor binding pair can beconjugated to the compounds provided herein for targeting the compoundsto specific target tissues or target compositions. Examples ofligand-receptor binding pairs are set out in U.S. Pat. Nos. 4,374,925and 3,817,837, the teachings of which are incorporated herein byreference.

Conjugation to ligands. Many compounds that can serve as targets forligand-receptor binding pairs, and more specifically, antibodies, havebeen identified, and the techniques to construct conjugates of suchligands with photosensitizers are well known to those of ordinary skillin this art. For example, Rakestraw et al. teaches conjugating Sn(IV)chlorin e via covalent bonds to monoclonal antibodies using a modifieddextran carrier (Rakestraw, S. L., Tompkins, R. D., and Yarmush, M. L.,Proc. Nad. Acad. Sci. USA 87: 4217-4221 (1990). The compounds disclosedherein can also be conjugated to a ligand, such as an antibody, by usinga coupling agent. Any bond which is capable of linking the componentssuch that they are stable under physiological conditions for the timeneeded for administration and treatment is suitable, but covalentlinkages are preferred. The link between two components may be direct,e.g., where a photosensitizer is linked directly to a targeting agent,or indirect, e.g., where a photosensitizer is linked to an intermediateand that intermediate being linked to the targeting agent.

A coupling agent should function under conditions of temperature, pH,salt, solvent system, and other reactants that substantially retain thechemical stability of the photosensitizer, the backbone (if present),and the targeting agent. Coupling agents should link component moietiesstably, but such that there is only minimal or no denaturation ordeactivation of the photosensitizer or the targeting agent. Manycoupling agents react with an amine and a carboxylate, to form an amide,or an alcohol and a carboxylate to form an ester. Coupling agents areknown in the art (see, e.g., M. Bodansky, “Principles of PeptideSynthesis”, 2nd ed., and T. Greene and P. Wuts, “Protective Groups inOrganic Synthesis,” 2nd Ed, 1991, John Wiley, NY).

The conjugates of the compounds provided herein with ligands such asantibodies can be prepared by coupling the compound to targetingmoieties by cleaving the ester on the “d” ring and coupling the compoundvia peptide linkages to the antibody through an N terminus, or by othermethods known in the art. A variety of coupling agents, includingcross-linking agents, can be used for covalent conjugation. Examples ofcross-linking agents include N,N′-dicyclohexylcarbodiimide (DCC),N-succinimidyl-S-acetyl-thioacetate (SATA),N-succinimidyl-3-(2-pyridyidi-thio)propionate (SPDP),ortho-phenylene-dimaleimide (o-PDM), and sulfosuccinimidyl4-(N-maleimido-methyl)-cyclohexane-1-carboxylate (sulfo-SMCC). See,e.g., Karpovsky et al. J. Exp. Med. 160:1686 (1984); and Liu, M A etal., Proc. Natl. Acad. Sci. USA 82: 8648 (1985). Other methods includethose described by Brennan et al. Science 229: 81-83 (1985) and Glennieet al., J. Immunol. 139: 2367-2375 (1987). A large number of couplingagents for peptides and proteins, along with buffers, solvents, andmethods of use, are described in the Pierce Chemical Co. catalog, pagesO-90 to O-110 (1995, Pierce Chemical Co., 3747 N. Meridian Rd., RockfordIll., 61105, U.S.A.), which catalog is hereby incorporated by reference.

For example, DCC is a useful coupling agent that can be used to promotecoupling of the alcohol NHS to chlorin e6 in DMSO forming an activatedester which can be cross-linked to polylysine. DCC is a carboxy-reactivecross-linker commonly used as a coupling agent in peptide synthesis, andhas a molecular weight of 206.32. Another useful cross-linking agent isSPDP, a heterobifunctional cross-linker for use with primary amines andsulfhydryl groups. SPDP has a molecular weight of 312.4, a spacer armlength of 6.8 angstroms, is reactive to NHS-esters and pyridyldithiogroups, and produces cleavable cross-linking such that, upon furtherreaction, the agent is eliminated so the photosensitizer can be linkeddirectly to a backbone or targeting agent. Other useful conjugatingagents are SATA for introduction of blocked SH groups for two-stepcross-linking, which is deblocked with hydroxylamine-HCl, andsulfo-SMCC, reactive towards amines and sulfhydryls. Other cross-linkingand coupling agents are also available from Pierce Chemical Co.Additional compounds and processes, particularly those involving aSchiff base as an intermediate, for conjugation of proteins to otherproteins or to other compositions, for example to reporter groups or tochelators for metal ion labeling of a protein, are disclosed in EPO243,929 A2 (published Nov. 4, 1987).

Photosensitizers which contain carboxyl groups can be joined to lysineε-amino groups in the target polypeptides either by preformed reactiveesters (such as N-hydroxy succinimide ester) or esters conjugated insitu by a carbodiimide-mediated reaction. The same applies tophotosensitizers which contain sulfonic acid groups, which can betransformed to sulfonyl chlorides which react with amino groups.Photosensitizers which have carboxyl groups can be joined to aminogroups on the polypeptide by an in situ carbodiimide method.Photosensitizers can also be attached to hydroxyl groups, of serine orthreonine residues or to sulfhydryl groups of cysteine residues.

Methods of joining components of a conjugate, e.g., coupling polyaminoacid chains bearing photosensitizers to antibacterial polypeptides, canuse heterobifunctional cross linking reagents. These agents bind afunctional group in one chain and to a different functional group in thesecond chain. These functional groups typically are amino, carboxyl,sulfhydryl, and aldehyde. There are many permutations of appropriatemoieties which will react with these groups and with differentlyformulated structures, to conjugate them together. See the PierceCatalog, and Merrifield, R. B. et al., Ciba Found Symp. 186: 5-20(1994).

The compounds or pharmaceutically acceptable derivatives thereof may bepackaged as articles of manufacture containing packaging material, acompound or pharmaceutically acceptable derivative thereof providedherein, which is effective for modulating the activity ofhyperproliferating tissue or neovascularization, or for treatment,prevention or amelioration of one or more symptoms of hyperproliferatingtissue or neovascularization mediated diseases or disorders, or diseasesor disorders in which hyperproliferating tissue or neovascularizationactivity, is implicated, within the packaging material, and a label thatindicates that the compound or composition, or pharmaceuticallyacceptable derivative thereof, is used for modulating the activity ofhyperproliferating tissue or neovascularization, or for treatment,prevention or amelioration of one or more symptoms of hyperproliferatingtissue or neovascularization mediated diseases or disorders, or diseasesor disorders in which hyperproliferating tissue or neovascularization isimplicated.

The articles of manufacture provided herein contain packaging materials.Packaging materials for use in packaging pharmaceutical products arewell known to those of skill in the art. See, e.g., U.S. Pat. Nos.5,323,907; 5,052,558 and 5,033,252. Examples of pharmaceutical packagingmaterials include, but are not limited to, blister packs, bottles,tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, andany packaging material suitable for a selected formulation and intendedmode of administration and treatment. A wide array of formulations ofthe compounds and compositions provided herein are contemplated as are avariety of treatments for any disease or disorder in whichhyperproliferating tissue or neovascularization is implicated as amediator or contributor to the symptoms or cause.

3. Methods of Use.

A. Methods of PDT, Diagnostic and Therapeutic Applications. Briefly, thephotosensitizing compound is generally administered to the subjectbefore the target tissue, target composition or subject is subjected toillumination. The photosensitizing compound is administered as describedelsewhere herein.

The dose of photosensitizing compound can be determined clinically.Depending on the photosensitizing compound used, an equivalent optimaltherapeutic level will have to be established. A certain length of timeis allowed to pass for the circulating or locally deliveredphotosensitizer to be taken up by the target tissue. The unboundphotosensitizer is cleared from the circulation during this waitingperiod, or additional time can optionally be provided for clearing ofthe unbound compound from non-target tissue. The waiting period will bedetermined clinically and may vary from compound to compound.

At the conclusion of this waiting period, a laser light source or anon-laser light source (including but not limited to artificial lightsources such as fluorescent or incandescent light, or natural lightsources such as ambient sunlight) is used to activate the bound drug.The area of illumination is determined by the location and dimension ofthe pathologic region to be detected, diagnosed or treated. The durationof illumination period will depend on whether detection or treatment isbeing performed, and can be determined empirically. A total orcumulative period of time anywhere from between about 4 minutes and 72hours can be used. In one embodiment, the illumination period is betweenabout 60 minutes and 148 hours. In another embodiment, the illuminationperiod is between about 2 hours and 24 hours.

Preferably, the total fluence or energy of the light used forirradiating, as measured in Joules, is between about 10 Joules and about25,000 Joules; more preferably, between about 100 Joules and about20,000 Joules; and most preferably, between about 500 Joules and about10,000 Joules. Light of a wavelength and fluence sufficient to producethe desired effect is selected, whether for detection by fluorescence orfor therapeutic treatment to destroy or impair a target tissue or targetcomposition. Light having a wavelength corresponding at least in partwith the characteristic light absorption wavelength of thephotosensitizing agent is preferably used for irradiating the targetissue.

The intensity or power of the light used is measured in watts, with eachJoule equal to one watt-sec. Therefore, the intensity of the light usedfor irradiating in the present invention may be substantially less than500 mW/cm². Since the total fluence or amount of energy of the light inJoules is divided by the duration of total exposure time in seconds, thelonger the amount of time the target is exposed to the irradiation, thegreater the amount of total energy or fluence may be used withoutincreasing the amount of the intensity of the light used. The presentinvention employs an amount of total fluence of irradiation that issufficiently high to activate the photosensitizing agent.

In one embodiment of using compounds disclosed herein for photodynamictherapy, the compounds are injected into the mammal, e.g. human, to bediagnosed or treated. The level of injection is usually between about0.1 and about 0.5 umol/kg of body weight. In the case of treatment, thearea to be treated is exposed to light at the desired wavelength andenergy, e.g. from about 10 to 200 J/cm². In the case of detection,fluorescence is determined upon exposure to light at a wavelengthsufficient to cause the compound to fluoresce at a wavelength differentthan that used to illuminate the compound. The energy used in detectionis sufficient to cause fluorescence and is usually significantly lowerthan is required for treatment.

Any one of the photosensitizing compounds disclosed herein or apharmaceutically acceptable derivative thereof may be supplied in a kitalong with instructions on conducting any of the methods disclosedherein. Instructions may be in any tangible form, such as printed paper,a computer disk that instructs a person how to conduct the method, avideo cassette containing instructions on how to conduct the method, orcomputer memory that receives data from a remote location andillustrates or otherwise provides the instructions to a person (such asover the Internet). A person may be instructed in how to use the kitusing any of the instructions above or by receiving instructions in aclassroom or in the course of treating a patient using any of themethods disclosed herein, for example.

Additional examples and specific examples of methods of using compoundsand compositions of the present invention include but are not limited tothe following:

(i) Treatment of opportunistic infections. Compounds, compositions andmethods of the invention are useful for PDT of opportunistic infections,particularly of soft tissue. For antimicrobial treatment (via PDT) ofinfections, particularly wound infections, the infecting organism caninclude (as non limiting examples) Staphylococcus aureus, Pseudomonasaeruginosa, Escherichia coli. In nosocomial infections, P. aeruginosa isresponsible for 8% of surgical-wound infections and 10% of bloodstreaminfections. In some embodiments the subjects are immunocompromisedsubjects, such as those afflicted with AIDS or undergoing treatment withimmunosupressive agents.

(ii) Treatment of burns. Infections by S. aureus and gram-positivebacteria in general are particularly pronounced in burns (Lambrechts,2005). The multidrug resistance of S. aureus presents significantmedical challenges. In this regard, compounds, compositions and methodsof the invention are useful for the treatment of opportunisticinfections of burns.

(iii) Sepsis. Compounds, compositions and methods of the invention areuseful for the PDT treatment of subjects afflicted with opportunisticinfections of Vibrio vulnificus. V. vulnificus, a gram-negativebacterium, causes primary sepsis, wound infections, and gastrointestinalillness in humans.

(iv) Ulcers. Compounds, compositions and methods of the invention areuseful for PDT treatment of the bacterium that causes ulcers(Helicobacter pylori). In the clinic, treatment can be effected in anysuitable manner, such as by insertion of a fiber optic cable (akin to anendoscope but with provisions for delivery of red or near-IR light) intothe stomach or afflicted region.

(v) Periodontal disease. Compounds, compositions and methods of theinvention are useful in PDT for the treatment of periodontal disease,including gingivitis. Periodontal disease is caused by the overgrowth ofbacteria, such as the gram-negative anaerobe Porphyromonas gingivalis.As with many PDT treatments, targeting or solubilizing entities inconjunction with the photoactive species are essential for appropriatedelivery of the photoactive species to the desired cells. The oralpathogens of interest for targeting include Porphyromonas gingivalis,Actinobacillus actinomycetemcomitans, Bacteroides forsythus,Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatumsubsp. Polymorphum, Actinomyces viscosus, and the streptococci. For suchapplications the compounds or compositions of the invention can betopically applied (e.g., as a mouthwash or rinse) and then lightadministered with an external device, in-the-mouth instrument, orcombination thereof.

(vi) Atherosclerosis. Compounds, compositions and methods of theinvention are useful in PDT to treat vulnerable atherosclerotic plaque.Without wishing to be bound to any particular theory, invadinginflammatory macrophages are believed to secrete metalloproteinases thatdegrade a thin layer of collagen in the coronary arteries, resulting inthrombosis, which often is lethal (Demidova and Hamblin, 2004).Bacteriochlorins targeted to such inflammatory macrophages are usefulfor PDT of vulnerable plaque.

(vii) Cosmetic and dermatologic applications. Compounds, compositionsand methods of the invention are useful in PDT to treat a wide range ofcosmetic dermatological problems, such as hair removal, treatment ofpsoriasis, or removal of skin discoloration. Ruby lasers are currentlyused for hair removal; in many laser treatments melanin is thephotosensitized chromophore. Such treatments work reasonably well forfair-skinned individuals with dark hair. Compounds, compositions andmethods of the invention can be used as near-IR sensitizers for hairremoval, which enables targeting a chromophore with a more specific andsharp absorption band.

(viii) Acne. Compounds, compositions and methods of the invention areuseful in PDT to treat acne. Acne vulgaris is caused byPropionibacterium acnes, which infects the sebaceous gland; some 80% ofyoung people are affected. Here again, the growing resistance ofbacteria to antibiotic treatment is leading to an upsurge of acne thatis difficult to treat. Current PDT treatments of acne typically rely onthe addition of aminolevulinic acid, which in the hair follicle orsebaceous gland is converted to free base porphyrins. Compounds andcompositions of the invention can be administered to subjects topicallyor parenterally (e.g., by subcutaneous injection) depending upon theparticular condition.

(ix) Infectious diseases. Compounds, compositions and methods of theinvention are useful in PDT to treat infectious diseases. For example,Cutaneous leishmaniasis and sub-cutaneous leishmaniasis, which occursextensively in the Mediterranean and Mideast regions, is currentlytreated with arsenic-containing compounds. PDT has been used toreasonable effect recently, at least in one case, on a human patient.The use of compounds and compositions of the present invention arelikewise useful, and potentially offer advantages such as ease ofsynthesis and better spectral absorption properties.

(x) Tissue sealants. Compounds, compositions and methods of theinvention are useful in PDT as tissue sealants in subjects in needthereof. Light-activated tissue sealants are attractive for sealingwounds, bonding tissue, and closing defects in tissue There are manyapplications where sutures or staples are undesirable, and use of suchmechanical methods of sealing often lead to infection and scarring.

(xi) Neoplastic disease. Compounds, compositions and methods of theinvention are useful in PDT for treating neoplastic diseases or cancers,including skin cancer, lung cancer, colon cancer, breast cancer,prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma,leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque-stagecutaneous T-cell lymphoma, and Kaposi sarcoma.

B. Imaging Enhancing Agents. In addition to PDT, the compositionsprovided herein can be used as imaging enhancing agents in diagnosticimaging techniques, or for the labeling of target tissues or targetcompositions for diagnostic radiology. In the modern medical field,there are a variety of treatments including magnetic resonance imaging(MRI) for the diagnosis of diseases. Detection of cancer in its earlystages should improve the ability to cure eliminate the canceroustissue. Early diagnosis of precancerous regions and minute cancer areimportant subject matters in modern cancer treatments. MRI has emergedas a powerful tool in clinical settings because it is noninvasive andyields an accurate volume rendering of the subject. The image is createdby imposing one or more orthogonal magnetic field gradients upon thesubject or specimen while exciting nuclear spins with radio frequencypulses as in a typical nuclear magnetic resonance (NMR) experiment.After collection of data with a variety of gradient fields,deconvolusion yields a one, two, or three dimensional image of thespecimen/subject. Typically, the image is based on the NMR signal fromthe protons of water where the signal intensity in a given volumeelement is a function of the water concentration and relaxation times.Local variation in there parameters provide the vivid contrast observedin MR images.

MRI contrast agents act by increasing the rate of relaxation, therebyincreasing the contrast between water molecules in the region where theimaging agent accretes and water molecules elsewhere in the body.However, the effect of the agent is to decrease both T₁ and T₂, theformer resulting in greater contrast while the latter results in lessercontrast. Accordingly, the phenomenon is concentration-dependent, andthere is normally an optimum concentration of a paramagnetic species formaximum efficacy. This optimal concentration will vary with theparticular agent used, the locus of imaging, the mode of imaging, i.e.,spin-echo, saturation-recovery, inversion-recovery and/or various otherstrongly T₁-dependent or T₂-dependent imaging techniques, and thecomposition of the medium in which the agent is dissolved or suspended.These factors, and their relative importance are known in the art. See,e.g., Pykett, Scientific American 246: 78 (1982); Runge et al., Am. J.Radiol. 141: 1209 (1983). When MRI contrast agents are useddiagnostically, they are vascularly perfused, enhancing the contrast ofblood vessels and reporting on organ lesions and infiltration. However,the labeling of specific tissues for diagnostic radiology remains adifficult challenge for MRL Efforts to develop cell and tissue-specificMRI image enhancing agents by modifying existing immunologicaltechniques has been the focus of much research in diagnostic radiology.For example, antibodies labeled with paramagnetic ions, generally thegadolinium chelate Gd-DTPA, have been generated and tested for theireffects on MRI contrast of tumors and other tissues (U.S. Pat. No.5,059,415). Unfortunately, the relaxivity of Gd bound to antibodies hasbeen found to be only slightly better than that of unbound Gd-DTPA(Paajanen et al., Magn. Reson. Med 13: 38-43 (1990)).

MRI is generally used to detect ¹H nuclei in the living body. However,MRI is capable of detecting NMR spectrums of other nuclear species,including ¹³C, ¹⁵N, ³¹P, and ¹⁹F. The ¹⁹F is not abundant in the livingbody. By incorporating isotopes useful in MRI, such as ¹³C, ¹⁵N, ³¹P, or¹⁹F, and particularly ¹⁹F in the compositions provided herein andadministering to a subject, the compounds provided herein wouldaccumulate in target tissue, and subsequent MR imaging would produce NMRdata with enhanced signal from the targeted tissue or targetcompositions due to the presence of the accumulated compound with theMRI recognizable isotope, such as ¹⁹F. Thus, the disclosed compounds canbe used as image enhancing agents and provide labeling of specifictarget tissues or target compositions for diagnostic radiology,including MRI.

C. Detecting Target Tissue or Target Compositions. In addition to PDT,the compositions provided herein can be used to detect target cells,target tissue, or target compositions in a subject. When the compoundsprovided herein are to be used for detection of target tissue or targetcomposition, the compounds are introduced into the subject andsufficient time is allowed for the compounds to accumulate in the targettissue or to become associated with the target composition. The area oftreatment is then irradiated, generally using light of an energysufficient to cause fluorescence of the compound, and the energy used isusually significantly lower than is required for photodynamic therapytreatment. Fluorescence is determined upon exposure to light at thedesired wavelength, and the amount of fluorescence can be correlated tothe presence of the compound, qualitatively or quantitatively, bymethods known in the art.

D. Diagnosing an Infecting Agent. The compositions provided herein canbe used to diagnose the presence of an infecting agent, or the identityof an infecting agent in a subject. The compounds provided herein can beconjugated to one or more ligands specific for an infecting agent, suchas an antibody or antibody fragment, that selectively associates withthe infecting agent, and after allowing sufficient time for the targetedcompound to associate with the infecting agent and to clear fromnon-target tissue, the compound can be visualized, such as by exposingto light of an energy sufficient to cause fluorescence of the compound,or by imaging using diagnostic radiology, including MRI. By way ofexample, any one of the compounds provided herein can be conjugated toan antibody that is targeted against a suitable Helicobacter pyloriantigen, and formulated into a pharmaceutical preparation that, whenintroduced into a subject, releases the conjugated compound to a gastricmucus/epithelial layer where the bacterium is found. After sufficienttime for the compound to selectively associate with the target infectingagent, and for any unbound compound to clear from non-target tissue, thesubject can be examined to determine whether any Helicobacter pylori ispresent. This can be done by MRI to detect accumulated compound becauseof the presence of ¹⁹F substituents, for example, or by irradiating thesuspect target area with light of an energy sufficient to causefluorescence of the compound, such as by using fiberoptics, anddetecting any fluorescence of the targeted compound.

4. Solar Cells, Light Harvesting Rods and Light Harvesting Arrays.

Bacteriochlorins of Formula I herein may be used as chromophores (alsoreferred to as photosensitizers or simply sensitizers) in solar cells,including but not limited to high surface area colloidal semiconductorfilm solar cells (Gratzel cells), as described in, for example, U.S.Pat. Nos. 5,441,827; 6,420,648; 6,933,436; 6,924,427; 6,913,713;6,900,382; 6,858,158; and 6,706,963.

Bacteriochlorins of Formula I may be used as chromophores in the lightharvesting rods described in U.S. Pat. Nos. 6,407,330 and 6,420,648(incorporated herein by reference). The light harvesting rod maycomprise one or more bacteriochlorins of Formula I coupled to one or twoadjacent chromophores depending upon the position thereof in the lightharvesting rod. Such light harvesting rods may be utilized to producelight harvesting arrays as described in U.S. Pat. No. 6,420,648 andsolar cells as described in U.S. Pat. No. 6,407,330.

5. Flow Cytometry.

Flow cytometry is known and described in, for example, U.S. Pat. Nos.5,167; 5,915,925; 6,248,590; 6,589,792; and 6,890,487. In someembodiments the particle being detected, such as a cell, is labelledwith a luminescent compound such as a phosphor or fluorophore fordetection. Labelling can be carried out by any suitable technique suchas coupling the luminescent compound to another compound such as anantibody which in turn specifically binds to the particle or cell, byuptake or internalization of the luminescent compound into the cell orparticle, by non-specific adsorption of the luminescent compound to thecell or particle, etc. The bacteriochlorins described herein are usefulin flow cytometry as such luminescent compounds, which flow cytometrytechniques (including fluorescent activated cell sorting or FACS) may becarried out in accordance with known techniques or variations thereofwhich will be apparent to those skilled in the art based upon theinstant disclosure.

5. Information Storage Devices.

Bacteriochlorins of the invention are also useful immobilized to asubstrate for making charge storage molecules and information storagedevices containing the same, either individually or as linked polymersthereof, either optionally including additional compounds to addadditional oxidation states. Such charge storage molecules andinformation storage devices are known and described in, for example,U.S. Pat. No. 6,208,553 to Gryko et al.; U.S. Pat. No. 6,381,169 toBocian et al.; and U.S. Pat. No. 6,324,091 to Gryko et al. Thebacteriochlorins of the invention may comprise a member of a sandwichcoordination compound in the information storage molecule, such asdescribed in U.S. Pat. No. 6,212,093 to Li et al. or U.S. Pat. No.6,451,942 to Li et al.

The present invention is explained in greater detail in the followingnon-limiting Examples.

Experimental

The strong absorption by bacteriochlorins in the near-IR region makesthese pigments especially attractive for a wide variety of applicationsin medicine, the life sciences, and materials chemistry. Suchapplications require simple synthetic methods that afford access toample quantities of stable compounds. Specific attributes of the desiredmethodology include the ability (1) to construct a macrocycle that islocked at the bacteriochlorin reduction level, and (2) to exercisesynthetic control over the pattern of substituents arrayed around theperimeter of the macrocycle. The former objective can be met through theuse of a geminal dimethyl group in each of the two pyrroline rings. Thelatter objective can be met by using hydrodipyrrin precursors containingthe desired pattern of substituents to be carried over to the targetbacteriochlorin. The structure of a target bacteriochlorin core (lackingperipheral substituents other than the geminal dimethyl groups) is shownin Chart 3.

We here describe a de novo synthesis of bacteriochlorins where thebacteriochlorin reduction level is established by structural features inacyclic precursors to the macrocycle. The route that we developed drawson our prior work concerning the rational syntheses of chlorins(Strachan, J.-P. et al., J. Org. Chem. 2000, 65, 3160-3172; Strachan,J.-P. et al., J. Org. Chem. 2000, 65, 3160-3172) but also requiredextensive development. The route to bacteriochlorins employs theself-condensation of a dihydrodipyrrin-acetal wherein the pyrrolic endof the molecule functions as a nucleophile and the acetal functions asan electrophile. We describe the synthesis of thedihydrodipyrrin-acetal, a study of conditions for the self-condensationof the dihydrodipyrrin-acetal, and the characterization of thebacteriochlorins.

I. Experimental Section.

General. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were collectedat room temperature in CDCl₃. Absorption spectra were collectedroutinely. Melting points are uncorrected. Column chromatography wasperformed with flash silica or alumina (80-200 mesh). Bacteriochlorinswere analyzed in neat form by laser desorption mass spectrometry (LD-MS)in the absence of a matrix. The CHCl₃ contained 0.8% ethanol.

Static Absorption and Emission. Static absorption and fluorescencemeasurements were performed as described previously (Li, F. et al., J.Mater. Chem. 1997, 7, 1245-1262; Strachan, J.-P. et al., J. Am. Chem.Soc. 1997, 119, 11191-11201; Yang, S. I. et al., J. Phys. Chem. B 1998,102, 9426-9436). Non-deaerated samples with an absorbance ˜0.15 atλ_(exc) were used for the key emission measurements; the detectionband-pass was 4-5 nm and the spectra were corrected for thedetection-system spectral response.

Extinction Coefficients. The extinction coefficients of 12, 13, andIn-12 were determined by dissolving a known quantity of eachbacteriochlorin (˜6 mg) in 100 mL of toluene. Then a known amount (˜100μL) of this solution was added to a quartz cuvette containing 3.0 mL oftoluene. The absorption spectrum was recorded at room temperature.

Fluorescence Quantum Yields (Φ_(f)). The Φ_(f) values were determinedwith bacteriochlorin samples where the Q_(x)(0,0)-band absorptions [511(12) or 499 nm (13)] were ˜0.08-0.1. The reference compound (t-Bu)₄H₂Pc,which emits in the region (˜680-820) similar to that of thebacteriochlorins, was used as the standard (Φ_(f)=0.77) (Teuchner, K. etal., Photochem. Photobiol. 1993, 57, 465-471). The absorption (644 nm)of (t-Bu)₄H₂Pc was also between 0.08-0.1. Excitations were performed at511 or 499 nm and the fluorescence emission spectra were obtained(600-900 nm) with correction for instrument response and temporalvariation in light intensity. The spectra were then integrated from600-900 nm, affording values for I_(em). The I_(em) values were dividedby the absorption recorded at 511 or 499 nm respectively. Since theabsolute Φ_(f) for (t-Bu)₄H₂Pc is known (0.77), the term I_(em)/A₆₄₄obtained for (t-Bu)₄H₂Pc and for the bacteriochlorins can thus be usedto calculate the relative Φ_(f) for the bacteriochlorins. All data wereobtained in toluene at room temperature.

General procedure for investigation of conditions in bacteriochlorinforming reaction. The condensations were carried out in 1-dram vialscontaining magnetic stir bars. A freshly prepared sample ofdihydrodipyrrin-acetal 11 (0.85-1.7 mg) was dissolved in a specificamount (0.50-2.0 mL) of a certain solvent. The condensation wasinitiated by adding the desired acid to the stirred reaction mixture atroom temperature. The progress of the reaction was monitored by takingaliquots periodically from the reaction mixture via syringe andneutralizing with TEA, followed by absorption spectroscopy. Inparticular, for 5 mM reactions of 11, 10 μL aliquots were removed fromthe reaction vessel and diluted with 3 mL of CH₂Cl₂. The dilutedsolution was treated with one drop of TEA and the visible absorptionspectrum was recorded. [In cases where the acid yielded a heterogeneousmixture (e.g., InCl₃, Yb(OTf)₃) or broadened absorption in the Q_(y)region, the 10 μL aliquots were passed through a 2-cm long pipet column(CH₂Cl₂/ethyl acetate). The first collected sample (green; eluted withCH₂Cl₂) and the second collected sample (pink; eluted with ethylacetate) were separately concentrated and then diluted with 3 mL ofCH₂Cl₂. The visible absorption spectrum was recorded with the dilutedsolutions.] The yield of bacteriochlorins was determined by theintensity of the Q_(y) band (above 700 nm, ε=120,000 M⁻¹cm⁻¹) measuredfrom the apex to the middle point of base line, which lined from theblue edge to the red edge of the band. This eliminated the contributionof the other components, which may have absorption band in the region ofabove 700 nm.

In the case of insoluble acids, the dihydrodipyrrin-acetal 11 and theinsoluble acids were pre-weighed in the vial followed by addition of amicrostir bar. The reactions were initiated by addition of the desiredsolvent. The reactions were monitored as described above.

1,1-Dimethoxy-4-methyl-3-penten-2-one (2). A mixture of mesityl oxide(1) (18.0 mL, 160 mmol), diphenyl diselenide (5.00 g, 16 0 mmol), andammonium peroxydisulfate (109 g, 480 mmol) in anhydrous MeOH (1.20 L)was refluxed for 4 h under argon. The progress of the reaction wasmonitored by TLC. The reaction mixture was poured into water (1.20 L)and extracted with chloroform. The organic layer was washed with water,dried (Na₂SO₄), and concentrated to give a dark brown oil. Bulb-to-bulbdistillation of the oil at 50° C./0.04-0.05 mmHg gave a yellow oil. Theoil was chromatographed [silica, hexanes/ethyl acetate (3:1)] to give apale yellow oil (7.37 g, 29%). Analytical data were consistent with theliterature¹² for the title compound: ¹H NMR δ 1.96 (d, J=1.2 Hz, 3H),2.21 (d, J=1.2 Hz, 3H), 3.42 (s, 6H), 4.49 (s, 1H), 6.36-6.38 (m, 1H);¹³C NMR δ 21.3, 28.2, 54.5, 104.5, 119.1, 160.2, 194.2; FAB-MS obsd159.1020, calcd 159.1021 (C₈H₁₄O₃) [M+H]⁺. Note: The use of reagentgrade methanol resulted in a slow reaction (required >26 h forcompletion) versus the relatively fast reaction (<4 h) when anhydrousmethanol was used.

In situ generation of mono-Ethyl malonate (4). A solution of ethylmalonate potassium salt (27.0 g, 158 mmol) in water (20.0 mL) wastreated with concentrated HCl (˜35%, 15.0 mL) and the resulting mixturewas stirred for 10 min at room temperature. The mixture was extractedwith ether. The extracts were washed with water, dried (Na₂SO₄), andconcentrated to give a colorless oil (16.7 g, 79%). The oil was used forsubsequent reaction without characterization.

Ethyl-3-(4-methylphenyl)prop-2-enoate (5). A solution of 3 (11.6 g, 96.9mmol) and 4 (16.7 g, 126 mmol) in piperidine (958 μL, 9.69 mmol) andpyridine (39.2 mL, 485 mmol) was refluxed for 8 h under argon. Thereaction mixture was cooled to room temperature and the reaction wasquenched with 2 N HCl (˜250 mL). The reaction mixture was extracted withether. The extracts were washed with water, base (NaHCO₃), and water.The organic solution was dried (Na₂SO₄), concentrated, andchromatographed (silica, CH₂Cl₂) to give a colorless oil (14.6 g, 79%):¹H NMR δ 1.33 (t, J=7.2 Hz, 3H), 2.37 (s, 3H), 4.26 (q, J=7.2 Hz, 2H),6.39 (d, J=15.8 Hz, 1H), 7.18 (d, J=8.2 Hz, 2H), 7.42 (d, J=8.2 Hz, 2H),7.66 (d, J=15.8 Hz, 1H); ¹³C NMR δ 14.5 21.6, 60.6, 117.4, 128.2, 129.8,131.9, 140.8, 144.8, 167.4; Anal. Calcd for C₁₂H₁₄O₂: C, 75.76; H, 7.42.Found: C, 75.76; H, 7.44.

3-(Ethoxycarbonyl)-4-(4-methylphenyl)pyrrole (6). A solution of TosMIC(15.7 g, 80.5 mmol) and 5 (14.6 g, 76.7 mmol) in a dry ether/DMSO (2:1)(154 mL) solution was added dropwise under argon to a stirred solutionof NaH (2.39 g, 99.7 mmol) in ether (70 mL). The mixture started toreflux due to the exothermic reaction. After 3 h, water (200 mL) wascarefully added to the mixture and the aqueous phase was extracted withether and CH₂Cl₂. The combined organic extracts were dried (Na₂SO₄),concentrated, and chromatographed [silica, CH₂Cl₂/ethyl acetate (9:1)]to give a light brown solid (13.1 g, 74%): mp 154-155° C.; ¹H NMR δ 1.25(t, J=7.2 Hz, 3H), 2.36 (s, 3H), 4.22 (q, J=7.2 Hz, 2H), 6.75-6.77 (m,1H), 7.16 (d, J=7.8 Hz, 2H), 7.38 (d, J=7.8 Hz, 2H), 7.46-7.48 (m, 1H),8.38-8.54 (br, 1H); ¹³C NMR δ 14.5, 21.4, 59.8, 113.9, 118.3, 125.4,126.8, 128.6, 129.4, 132.0, 136.3, 165.2; Anal. Calcd for C₁₄H₁₅NO₂: C,73.34; H, 6.59; N, 6.11. Found: C, 73.11; H, 6.59; N, 6.12.

3-(4-Methylphenyl)pyrrole (7). Following a standard procedure(Balasubramanian, T. et al., J. Org. Chem. 2000, 65, 7919-7929), amixture of 6 (6.81 g, 29.7 mmol) and ethylene glycol (76.0 mL) in a250-mL Claisen flask was flushed with argon for 10 min, and thenpowdered NaOH (3.05 g, 76.2 mmol) was added. The flask was placed in anoil bath at 120° C. and the oil bath temperature was raised to 160° C.After 2.5 h, the flask was cooled to room temperature and 10% aqueousNaCl (150 mL) was added. The aqueous layer was extracted with CH₂Cl₂.The organic layers were collected, washed with 10% aqueous NaCl, dried(Na₂SO₄), concentrated, and chromatographed (silica, CH₂Cl₂) to give alight brown solid (3.33 g, 71%): mp 92-93° C.; ¹H NMR δ 2.34 (s, 3H),6.51-6.54 (m, 1H), 6.82-6.84 (m, 1H), 7.05-7.08 (m, 1H), 7.15 (d, J=7.8Hz, 2H), 7.43 (d, J=7.8 Hz, 2H), 8.15-8.32 (br, 1H); ¹³C NMR δ 21.3,106.7, 114.4, 119.0, 125.1, 125.4, 129.5, 133.1, 135.2; FAB-MS obsd157.0885, calcd 157.0891 (C₁₁H₁₁N).

2-Formyl-3-(4-methylphenyl)pyrrole (8). Following a standard procedure(Balasubramanian, T. et al., J. Org. Chem. 2000, 65, 7919-7929), asolution of 7 (472 mg, 3.00 mmol) in DMF (0.96 mL) and CH₂Cl₂ (30 mL)under argon was cooled to 0° C. and then POCl₃ (340 μL, 3.60 mmol) wasadded dropwise. After 1 h, the flask was warmed to room temperature andstirred overnight (˜18 h). The reaction was quenched at 0° C. with 2.5 MNaOH (25 mL). The mixture was poured into water (50 mL), extracted withCH₂Cl₂, and the combined organic layers were washed with water, brine,dried (Na₂SO₄), and concentrated. The residue was chromatographed[silica, CH₂Cl₂/ethyl acetate (9:1)] to give a brown solid. ¹H NMRspectroscopy showed two regioisomers in ˜13:1 ratio. Cooling of thesolution (ethyl acetate/hexanes) at ˜−16° C. resulted in precipitationof an orange solid, which proved to be a single regioisomer (354 mg,64%): mp 149-150° C.; ¹H NMR δ 2.41 (s, 3H), 6.42-6.44 (m, 1H),7.10-7.13 (m, 1H), 7.26 (d, J=8.0 Hz, 2H), 7.40 (d, J=8.0 Hz, 2H),9.63-9.64 (m, 1H), 9.52-9.78 (br, 1H); ¹³C NMR δ 21.4, 111.6, 126.2,128.9, 129.3, 129.7, 130.9, 137.7, 137.9, 180.2; FAB-MS obsd 186.0907,calcd 186.0919 (C₁₂H₁₁NO).

2-(2-Nitroethyl)-3-(4-methylphenyl)pyrrole (9). A mixture of 8 (3.93 g,21.2 mmol), KOAc (2.29 g, 23.3 mmol), methylamine hydrochloride (1.72 g,25.4 mmol), and nitromethane (190 mL) under argon was stirred at roomtemperature. The mixture slowly became orange and yielded an orange-redprecipitate. After stirring for 2.5 h, TLC showed the appearance of anew component and the disappearance of 8. The reaction was quenched withbrine, extracted with ethyl acetate, and the organic layers were dried(Na₂SO₄) and concentrated. The residue was dissolved in THF/MeOH (210mL, 3:7) at 0° C. NaBH₄ (2.41 g, 63.6 mmol) was added in portions at 0°C. Then the mixture was stirred for 0.5 h at room temperature. Thereaction mixture was neutralized with acetic acid (pH=7), then water(150 mL) was added and the mixture was extracted with ethyl acetate. Thecombined organic layers were washed with water, brine, dried (Na₂SO₄),concentrated, and chromatographed [silica, hexanes/ethyl acetate (3:1)]to give a light brown solid (3.61 g, 74%): mp 81-82° C.; ¹H NMR δ 2.37(s, 3H), 3.44 (t, J=6.8 Hz, 2H), 4.54 (t, J=6.8 Hz, 2H), 6.27-6.29 (m,1H), 6.73-6.75 (m, 1H), 7.20 (d, J=8.2 Hz, 2H), 7.23 (d, J=8.2 Hz, 2H),8.19-8.36 (br, 1H); ¹³C NMR δ 21.2, 24.4, 75.2, 109.6, 117.7, 121.9,123.2, 128.0, 129.5, 133.4, 135.8; FAB-MS obsd 230.1055, calcd 230.1055(C₁₃H₁₄N₂O₂).

6-[3-(4-Methylphenyl)pyrrol-2-yl]-1,1-dimethoxy-4,4-dimethyl-5-nitro-2-hexanone(10). Following a general procedure (Strachan, J.-P et al., supra;Balasubramanian, T et al., supra), CsF (1.82 g, 12.0 mmol, 3.00 molequiv, freshly dried by heating to 100° C. under vacuum for 1 h and thencooling to room temperature under argon) was placed in a flask underargon. A mixture of 9 (921 mg, 4.00 mmol) and acetal 2 (6.33 g, 40.0mmol, 10 mol equiv) in dry acetonitrile (40 mL) was transferred bycannula to the flask containing CsF. The mixture was heated at 65° C.for 1.2 h, whereupon TLC analysis showed the reaction to be complete.The reaction mixture was filtered through a bed of silica (ethylacetate) and the filtrate was concentrated. The resulting oil wassubjected to bulb-to-bulb distillation at room temperature/0.04-0.05mmHg for 3 h, affording recovery of the acetal 2 (˜2 g) as thedistillate and the desired product in the crude undistilled residue.Purification of the residue by column chromatography [alumina, ethylacetate/hexanes (1:3)] gave a light brown solid (626 mg, 40%): mp98-100° C.; ¹H NMR δ 1.09 (s, 3H), 1.19 (s, 3H), 2.37 (s, 3H), 2.53,2.71 (AB, ²J=18.8 Hz, 2H), 3.21 (ABX, ³J=2.4 Hz, ²J=15.4 Hz, 1H), 3.39(ABX, ³J=11.6 Hz, ²J=15.4 Hz, 1H), 3.41 (s, 6H), 4.34 (s, 1H), 5.22(ABX, ³J=2.4 Hz, ³J=11.6 Hz, 1H), 6.22-6.24 (m, 1H), 6.66-6.68 (m, 1H),7.19 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H), 8.06-8.14 (br, 1H); ¹³CNMR δ 21.3, 24.1, 24.3, 25.3, 36.8, 45.1, 55.2, 55.2, 95.0, 104.7,109.5, 117.7, 122.1, 123.7, 128.4, 129.4, 133.5, 135.6, 203.7; Anal.Calcd for C₂₁H₂₈N₂O₅: C, 64.93; H, 7.27; N, 7.21. Found: C, 65.02; H,7.34; N, 7.14.

1-(1,1-Dimethoxymethyl)-3,3-dimethyl-7-(4-methylphenyl)-2,3-dihydrodipyrrin(11). Following the procedure for preparing a β-substituted pyrrole, asolution of 10 (237 mg, 0.610 mmol) in anhydrous THF (3.00 mL) underargon was treated with NaOMe (165 mg, 3.05 mmol) and the mixture wasstirred for 1 h at room temperature (first flask). In a second flask,TiCl₃ (8.6 wt % TiCl₃ in 28 wt % HCl, 4.56 mL, 3.05 mmol, 5.0 mol equiv)and H₂O (24 mL) were combined; NH₄OAc (18.8 g, 244 mmol) was added tobuffer the solution to pH 6.0; and then THF (1.60 mL) was added. Thesolution in the first flask containing the nitronate anion of 10 wastransferred via a cannula to the buffered TiCl₃ solution in the secondflask. The resulting mixture was stirred at room temperature for 6 hunder argon. Then the mixture was extracted with ethyl acetate. Thecombined organic layers were washed with 5% aqueous NaHCO₃ and water,and then dried (NaSO₄). The solvent was removed under reduced pressureat room temperature. The crude product was passed through a short column[alumina, hexanes/ethyl acetate (2:1)] to afford a light yellow solid(57 mg, 28%): mp 104-105° C.; ¹H NMR δ 1.19 (s, 6H), 2.38 (s, 3H), 2.62(s, 2H), 3.45 (s, 6H), 5.03 (s, 1H), 6.11 (s, 1H), 6.28-6.30 (m, 1H),6.86-6.88 (m, 1H), 7.22 (d, J=8.0 Hz, 2H), 7.35 (d, J=8.0 Hz, 2H),10.80-10.90 (br, 1H); ¹³C NMR δ 21.3, 29.3, 40.5, 48.3, 54.8, 103.0,106.2, 109.3, 119.2, 124.7, 126.9, 128.7, 129.4, 134.2, 135.4, 160.1,174.2; FAB-MS obsd 338.2020, calcd 338.1994; Anal. Calcd for C₂₁H₂₆N₂O₂:C, 74.52; H, 7.74; N, 8.28. Found: C, 74.46; H, 7.79; N, 8.08; λ_(abs)(CH₂Cl₂) 358.

7,8,17,18-Tetrahydro-5-methoxy-8,8,18,18-tetramethyl-2,12-bis(4-methlyphenyl)porphyrin(12). A solution of 11 (93 mg, 0.27 mmol) in CH₃CN (54 mL) was treatedwith neat BF₃.OEt₂ (350 μL, 2.7 mmol, 50 mM). The reaction mixture wasstirred at room temperature for 15 h. The reaction was monitored byabsorption spectroscopy. TEA (1.0 mL) was added to the reaction mixture.The reaction mixture was concentrated and the residue was dissolved inCH₂Cl₂. The solution was washed (water), dried (Na₂SO₄), concentratedand chromatographed [silica, CH₂Cl₂/hexanes (1:1)]. The first band(green) was collected (13, 8.1 mg, 11%) and then the second band (green)was collected (12, 24 mg, 30%). Data for 12: ¹H NMR δ −1.90 (br, 1H),−1.78 (br, 1H), 1.91 (s, 6H), 1.92 (s, 6H), 2.61 (s, 6H), 4.40 (s, 2H),4.41 (s, 2H), 4.49 (s, 3H), 7.58 (d, J=8.0 Hz, 4H), 8.10 (d, J=8.0 Hz,2H), 8.14 (d, J=8.0 Hz, 2H), 8.66-8.69 (m, 2H), 8.78 (s, 1H), 8.81 (s,1H), 8.94-8.95 (m, 1H); λ_(abs) (toluene)/nm 356 (c=110,000), 374(130,000), 511 (39,000), 732 (120,000 M⁻¹cm⁻¹); LD-MS obsd 580.1; FAB-MSobsd 580.3232, calcd 580.3202 (C₃₉H₄₀N₄O).

Data for 13: ¹H NMR δ −2.00 (br, 2H), 1.93 (s, 12H), 2.61 (s, 6H), 4.56(s, 4H), 7.59 (d, J=8.0 Hz, 4H), 8.13 (d, J=8.0 Hz, 4H), 8.73 (d, J=2.0Hz, 2H), 8.81 (s, 2H), 8.86 (s, 2H); λ_(abs) (toluene)/nm 351(ε=130,000), 374 (120,000), 499 (35,000), 737 (130,000 M⁻¹cm⁻¹); LD-MSobsd 550.0; FAB-MS obsd 550.3068, calcd 550.3096 (C₃₈H₃₈N₄).

II. Results and Discussion

Strategy. During the course of the development of a de novo synthesis ofbacteriochlorins, we examined the reactivity of a number ofhydrodipyrrins each containing one pyrrole and one pyrroline unit. Weeventually found that a dihydrodipyrrin (A) bearing a dimethyl acetalmoiety attached to the carbon of the pyrroline imine unit underwentself-condensation to give bacteriochlorins (Scheme 1).

This result validated the approach of employing pyrrole/pyrrolinemoieties to serve as complementary nucleophilic/electrophiliccounterparts. However, a number of problems with the condensation wereimmediately evident, including the very low yield (˜1%) and theformation of a mixture of bacteriochlorins. We decided to build on thisresult to develop a more reliable synthesis.

The initial target molecules of choice werehydrodipyrrin-carboxaldehydes rather than the acetal A. Thehydrodipyrrin-carboxaldehydes B and C differ only in the presence orabsence of an unsaturation at the 4,5-position (between the meso-carbonand the pyrroline α′-carbon). (Chart 4).

However, we were unable to prepare either B or C. Two observations fromthe literature concerning related structures (Chart 5) prompted the nextstep in the evolution of the synthesis.

First, Jacobi et al. developed a rational route to chlorins (Jacobi, P.A. et al., Org. Lett. 2001, 3, 831-834) wherein adiformyl-dihydrodipyrrin (D) was employed. The dihydrodipyrrin bears thedesired formyl group at the α-position of the pyrroline group, whilealso incorporating a nearly full complement of substituents throughoutthe molecule. Second, dihydrodipyrrins bearing an aryl substituent inthe β-position (E-a) of the pyrrole ring are known to be more stablethan unsubstituted analogs (E-b). Accordingly, we focused on thedevelopment of a route that would employ a β-pyrrole substituteddihydrodipyrrin analog (11) of the tetrahydrodipyrrin-acetal A. Thep-tolyl group was chosen as an inert substituent that is readilycharacterized by ¹H NMR spectroscopy.

Synthesis of Bacteriochlorin Precursors. The synthesis ofdihydrodipyrrin-acetal 11 was initiated in similar fashion to a priorsynthesis of a dihydrodipyrrin bearing a substituent at the β-positionof the pyrrole ring (E-a).

One of the key components in the synthesis of 11 is an α-keto acetal(2). Tiecco et al. reported the facile synthesis of α-keto acetals fromketones, including α-keto acetal 2,¹² albeit at a small scale. An excessamount of the α,β-unsaturated ketone (˜5-10 equiv) has typically beenemployed in Michael additions with a nitroethyl pyrrole. Thus, weapplied the method of Tiecco (Tiecco, M. et al., J. Org. Chem. 1990, 55,4523-4528) using a catalytic amount of diphenyl diselenide and excessammonium peroxydisulfate at much larger scale (160 mmol versus 2 mmol)to prepare α-keto acetal 2. Bulb-to-bulb distillation afforded a productthat showed unidentified impurities in the ¹H NMR spectrum. Subsequentchromatography gave pure α-keto acetal 2 (˜7 g) in 29% yield (Scheme 2).

Application of the previously unused Knoevenagel condensation (Silva, N.M. et al., Eur. J. Med. Chem. 2002, 37, 163-170; Diaz, J. L. et al.,Chem. Mater. 2002, 14, 2240-2251; Ren, X. et al., Tetrahedron: Asymmetry2002, 13, 1799-1804) of p-tolualdehyde with malonic acid monoethyl ester(Breslow, D. S. et al., J. Am. Chem. Soc. 1944, 66, 1286-1288) inpyridine containing a catalytic amount of piperidine gave the knownα,β-unsaturated ethyl cinnamate 5 (Tsuge, O. et al., J. Org. Chem. 1982,47, 5171-5177; Colas, C.; Goeldner, M. Eur. J. Org. Chem. 1999,1357-1366; Chuzel, O.; Piva, O. Synth. Commun. 2003, 33, 393-402) in 79%yield. Reaction of 5 with (p-tolylsulfonyl)methyl isocyanide (TosMIC)afforded β-substituted pyrrole 6 (a known compound with incomplete data(Di Santo, R. et al., Med. Chem. Res. 1997, 7, 98-108)) in 74% yield.Removal of the ethoxycarbonyl group of pyrrole 6 by treatment with NaOHin ethylene glycol at 160° C. gave the known β-substituted pyrrole 7(Sakai, K. et al., Chem. Pharm. Bull. 1980, 28, 2384-2393; Campi, E. M.et al., Aust. J. Chem. 1992, 45, 1167-1178; Pavri, N, P.; Trudell, M. L.J. Org. Chem. 1997, 62, 2649-2651) in 71% yield. Vilsmeier-Haackformylation of 7 yielded a mixture of regioisomers owing to substitutionat the 2- or 5-position. After column chromatography, the tworegioisomers were determined to be present in ˜13:1 ratio by ¹H NMRintegration of the methyl unit of the p-tolyl group. Selectiveprecipitation readily afforded the major regioisomer 8 in 64% yield(Scheme 3).

It is noteworthy that we previously employed the same formylation methodto prepare 2-formyl-3-(4-iodophenyl)pyrrole, which was characterized by¹H NMR spectroscopy and X-ray crystallography (Balasubramanian, T. etal., J. Org. Chem. 2000, 65, 7919-7929).^(E1) The chemical shift of thetwo peaks (δ 6.42-6.44 and 7.10-7.13 ppm) from the pyrrolic protons ofthe major isomer 8 were quite similar to those for2-formyl-3-(4-iodophenyl)pyrrole (8 6.42 and 7.14 ppm) and2-formyl-3-phenylpyrrole (8 6.50 and 7.30 ppm) (Cue, B. W. et al., J.Heterocycl. Chem. 1981, 18, 667-670). The minor isomers,2-formyl-4-(4-methylphenyl)pyrrole (δ 7.20-7.22 and 7.36-7.38 ppm) and2-formyl-4-(4-iodophenyl)pyrrole (δ 7.21 and 7.39 ppm), also showedsimilar chemical shifts for the respective pyrrolic protons.

Treatment of 8 to the standard conditions for nitro-aldol condensationwith pyrrole-2-carboxaldehyde (KOAc and a slight excess of methylaminehydrochloride in nitromethane at room temperature) for 2.5 h affordedthe crude nitrovinyl pyrrole as a brown solid. Reduction of the latterwith NaBH₄ gave the β-substituted nitroethylpyrrole 9 (74% for thistwo-step one-flask synthesis). The reaction of 9 with excess α-ketoacetal 2 (10 equiv) in the presence of CsF at 65° C. gave 10 in 40%yield, accompanied by recovery of acetal 2 (˜50%) upon bulb-to-bulbdistillation and column chromatography. Treatment of 10 with NaOMefollowed by a buffered TiCl₃ solution afforded the dihydrodiyrrin-acetal11 as a yellow solid in 28% yield.

Investigation of Reaction Conditions for Bacteriochlorin Formation. Aseries of microscale studies (˜1-2 mg of 11 per reaction) was performedto investigate effects of reaction parameters that are known toinfluence the course of condensations leading to porphyrinicmacrocycles, including concentration of 11, acid composition, acidconcentration, solvent, and time. The standard conditions employedinitially included 5 mM of acetal 11 in CH₃CN containing 50 mM of acidat room temperature, which closely resemble those in theself-condensation of the unsubstituted dihydrodipyrrin-acetal A. Sampleswere removed periodically over the course of ˜24 h, neutralized withTEA, and examined by absorption spectroscopy. Yields were calculated onthe basis of the assumption that each bacteriochlorin has ε_(Qy)=120,000M⁻¹cm⁻¹, an assumption that proved valid (vide infra).

A. Acids. A screening study was performed to identify the effects of avariety of acids on the self-condensation of 11. Four Brønsted acids andeleven Lewis acids were examined The acids can be categorized on thebasis of bacteriochlorin yields: BF₃.OEt₂ (31%); InCl₃, Sc(OTf)₃, orSnCl₄ (18-16%); p-TsOH.H₂O (4.9%); Yb(OTf)₃, SnF₄, TiCl₄, BBr₃, or HCl(1.5-0.4%), and AcOH, TFA, MgBr₂, ZnCl₂, or Zn(OAc)₂ (˜0%). The acidsInCl₃ and Yb(OTf)₃ gave a slightly different reaction course, affordinga free base bacteriochlorin, a metalated bacteriochlorin, and anon-bacteriochlorin macrocycle. This work will be described elsewhere.

B. Solvents. The effect of solvent on the self-condensation of 11 wasexamined with BF₃.OEt₂ catalysis under the standard conditions. Thebacteriochlorin yields were ˜30% (CH₃CN), <2% (CHCl₃ or ClCH₂CH₂Cl), andnot detectable (CH₂Cl₂, toluene, DMF, DMSO, THF, 1,4-dioxane, methanol,ethanol).

The reaction with BF₃.OEt₂ catalysis was scaled up. The reaction of 11(5 mM) with BF₃.OEt₂ (50 mM) in CH₃CN at room temperature proceededsmoothly and was complete in ˜5-6 h. Two bacteriochlorins [12 (30%), 13(11%)] were readily separated upon silica chromatography. Analysis by ¹HNMR spectroscopy showed that 12 has a methoxy group in a meso positionof the macrocycle whereas 13 is unsubstituted (Scheme 4). Eachbacteriochlorin was characterized by ¹H NMR, LD-MS, absorption spectra,and high resolution mass spectrometry (vide infra). The dramaticincrease in yield (versus the <1% yield with the unsubstituteddihydrodipyrrin-acetal A) validated our hunch that β-substitution wouldafford a more stable substrate and more efficient reaction.

C. Acid Concentration. The effect of BF₃.OEt₂ concentration on theself-condensation of 11 was examined next. Under the standard condition(5 mM of dihydrodipyrrin-acetal 11 in CH₃CN), the concentration ofBF₃.OEt₂ was examined over the range of 1-20 equivalents (i.e., 5-100mM). The yields of bacteriochlorins (sum of 12+13) are shown in FIG. 2.The reaction yields were quite dependent on the amount of acid used. Thecondensation of dihydrodipyrrin-acetal 11 generally proceeded wellwith >10 equiv of acid, while 20 equiv of BF₃.OEt₂ gave the highestoverall yield of the two bacteriochlorins.

Some information also was obtained concerning the ratio of products(12:13) as a function of acid concentration. For example when less than10 equiv of BF₃.OEt₂ was used, the major product was 12, whereas 20equiv of BF₃.OEt₂ gave 13 as a major product. The ratio of 12 to 13 wasnot determined because the respective absorption spectra are nearlyidentical, differing only by ˜10 nm in the Q_(x) bands [12 (511 nm), 13(499 nm)].

D. Concentration. The effect of different concentrations also wasexamined The concentration of dihydrodipyrrin-acetal 11 was examinedover the range 2.5-25 mM in CH₃CN with a constant 1:10 ratio ofconcentrations of 11 and BF₃.OEt₂ (i.e., the BF₃.OEt₂ concentration wasscaled from 25-250 mM). In general, higher concentrations affordedhigher yields of product. A further facet was that reaction at 10 mMgave 12 as a major product whereas that at 25 mM gave 13 as a majorproduct (FIG. 3).

Bacteriochlorin Formation. The studies described led to the followingreaction conditions. The reaction with dihydrodipyrrin-acetal 11 (5 mM)in CH₃CN containing BF₃.OEt₂ (50 mM) at room temperature gave twoseparable bacteriochlorins (12 and 13) in ˜40% overall yield (Scheme 4).The typical ratio of 12 and 13 was ˜3:1. Upon reaction at higherconcentrations of 11 (25 mM) and BF₃.OEt₂ (250 mM), 13 was the majorproduct with the total yield of both bacteriochlorins remaining ˜40%.These results show that the ratio of 12 and 13 can be influenced by theBF₃.OEt₂ concentration and the concentration of thedihydrodipyrrin-acetal 11.

The synthetic bacteriochlorins (12 and 13) are extraordinarily robust.For example, the bacteriochlorins are stable upon standing on the benchtop in solution open to air for more than 10 days, as well aschromatography on silica in air in the presence of bright ambientlighting. Unlike bacteriochlorins derived from photosynthetic bacteria,the synthetic bacteriochlorins do not undergo adventitiousdehydrogenation upon routine handling.

Mechanistic Considerations. At present we know very little about themechanistic course leading from the dihydrodipyrrin-acetal to thebacteriochlorin species. The balanced reaction for formation eachbacteriochlorin product (lacking aryl substituents for simplicity) isshown in Scheme 5.

The conversion of two molecules of the dihydrodipyrrin-acetal (DHDPA,analogous to 11) to give the 5-methoxybacteriochlorin (MeO-BC) mustproceed with elimination of three molecules of methanol. By contrast,the formation of the unsubstituted bacteriochlorin (H-BC) must proceedwith elimination of four molecules of methanol and addition of 2e- and2H⁺. Neither the source of the reductant nor the nature of theintermediate that undergoes reduction is known. It is worthwhile tocontrast this overall transformation with that of porphyrin formationfrom an aldehyde and pyrrole, which proceeds via condensation to give ahexahydroporphyrin (porphyrinogen) intermediate, which then is convertedvia a 6e-/6H⁺ oxidation to give the porphyrin (Lindsey, J. S. In ThePorphyrin Handbook; Kadish, K. M., Smith, K. M., Guilard, R., Eds.;Academic Press: San Diego, Calif., 2000; Vol. 1, pp 45-118).Bacteriochlorin formation from the dihydrodipyrrin-acetal does notrequire an oxidant. Further consideration of oxidation-state changes islikely to be important in searching for intermediates and in designingalternative precursors to give bacteriochlorins.

Characterization of the Bacteriochlorins. Absorption Spectra. Theabsorption spectra of 12 and 13 in toluene are shown in FIG. 4A. Freebase bacteriochlorin 12 exhibits broadened Soret bands with a singlepeak at 356 nm and a single peak at 374 nm. A weak Q_(x)(0,0) band and astrong Q_(y)(0,0) band appear at 511 and 732 nm, respectively. Free basebacteriochlorin 13 exhibits two Soret bands (B_(y), B_(x)) at 351 and374 nm. A weak Q_(x)(0,0) band and a strong Q_(y)(0,0) band appear at499 and 737 nm, respectively. The absorption spectrum of free basetetraphenylbacteriochlorin (H₂TPBC, λ_(abs) 356, 378, 520, and 742 nm)in benzene is very close to that of free base bacteriochlorins 12 and13. The overall spectral features and band intensities of thebacteriochlorins prepared herein resemble those of bacteriochlorophyll aas well as those of synthetic bacteriochlorins (Fajer, J. et al., Proc.Natl. Acad. Sci. USA. 1974, 71, 994-998; Hartwich, G. et al., J. Am.Chem. Soc. 1998, 120, 3675-3683; Whitlock, H. W. et al., J. Am. Chem.Soc. 1969, 91, 7485-7489; Dorough, G. D.; Miller, J. R. J. Am. Chem.Soc. 1952, 74, 6106-6108; Dorough, G. D.; Huennekens, F. M. J. Am. Chem.Soc. 1952, 74, 3974-3976; Miller, J. R.; Dorough, G. D. J. Am. Chem.Soc. 1952, 74, 3977-3981). The bacteriochlorins exhibit a light greenappearance in dilute solution in CH₂Cl₂ or toluene.

Fluorescence Properties. The fluorescence spectra and fluorescencequantum yields of 12 and 13 were collected in toluene at roomtemperature. The fluorescence spectrum of each bacteriochlorin isdominated by a Q_(y)(0,0) band with Stokes shift of 7-9 nm (FIG. 4B).The wavelength of the Q_(y)(0,0) emission maximum and fluorescencequantum yield (Φ_(f)) are given in Table 1. Measurements of thefluorescence quantum yield (Φ_(f)) of the bacteriochlorins using(t-Bu)₄H₂Pc (Φ_(f)=0.77) (Teuchner, K. et al., Photochem. Photobiol.1993, 57, 465-471) as a reference gave values of 0.21 and 0.22 for 12and 13, respectively.

Very few data are available concerning the fluorescence quantum yields(Φ_(f)) of bacteriochlorins. The free base analog of the naturallyoccurring bacteriochlorophyll a (bacteriopheophytin a) has Φ_(f)estimated to be 0.12, (Connolly, J. S. et al., Photochem. Photobiol.1982, 36, 565-574) whereas bacteriochlorin derivatives of13¹-deoxo-20-formyl-pyropheophorbide exhibited very low Φ_(f) values(˜0.002) (Pandey, R. K. et al., J. Med. Chem. 1997, 17, 2770-2779).Synthetic bacteriochlorins such asmeso-tetrakis(3-hydroxyphenyl)bacteriochlorin (Bonnett, R. et al., J.Chem. Soc. Perkin Trans. 2 1999, 325-328) or5,15-diphenylbacteriochlorin (Wang, T. Y. et al., Dyes and Pigments2002, 52, 199-208) gave Φ_(f) values of 0.11 or 0.14, respectively. Onthe other hand, the dioxobacteriochlorin derived from octaethylporphyrinwas reported to have Φ_(f) of 0.48. The dearth of fundamentalinformation concerning the fluorescence properties of bacteriochlorinsmust stem in large part to the prior lack of synthetic accessibility.

Laser Desorption Mass Spectrometry (LD-MS). Porphyrins typically give astrong molecule ion peak upon laser-desorption mass spectrometry (LD-MS)without requirement for use of a matrix.^(H8) LD-MS analysis of 12 or 13gave the molecule ion peak (m/z=580.1 or 550.0), consistent with theproposed structures (Scheme 4). The mass difference (˜30) between 12 and13 is consistent with the presence of the methoxy group in the formercompound.

¹H NMR Spectra. The ¹H NMR spectra of 12 and 13 are readily assignable.In the case of 13, which has C_(h) symmetry, a relatively simple ¹H NMRspectrum is observed. A broad upfield peak (δ −1.96 ppm), singlet at δ1.93 ppm, and singlet at δ 4.46 ppm are attributed to the two NHprotons, the pair of geminal dimethyl groups, and the CH₂ groups of thepyrroline rings, respectively. The aryl hydrogens of the p-tolyl groupsgive a characteristic pair of doublets (J=8.0 Hz) at δ 7.59 ppm and 8.13ppm. A doublet (J=2.0 Hz) at δ 8.73 ppm and two singlets at δ 8.81 and8.86 ppm stem from the six protons (3, 5, 10, 13, 15, and 20 positions)about the perimeter of the bacteriochlorin.

Bacteriochlorin 12 has generally similar features, but the presence ofthe 5-methoxy group results in C_(s) symmetry. Accordingly, the two NHprotons (δ −1.90 and −1.78 ppm), the two pairs of geminal dimethylgroups (δ 1.91 and 1.92 ppm), and the two methylene units in each of thereduced pyrrole rings (δ 4.40 and 4.41 ppm) are non-equivalent andappear as distinct singlets. Each p-tolyl group gives a pair of doublets(J=8.0, 8.4 Hz in each case) in the region of δ 7.56-7.59 ppm and8.09-8.15 ppm. The five peripheral protons (3, 10, 13, 15, and 20positions) give rise to apparent singlets (δ 8.68, 8.78, and 8.81 ppm),a doublet (δ 8.94 ppm), and a partially overlapping peak at δ 8.67 ppm.Taken together, the absorption, LD-MS, and ¹H NMR spectroscopic datasupport the structures proposed for bacteriochlorins 12 and 13.

Conclusions. A straightforward 8-step synthesis has been developed thataffords free base bacteriochlorins. Each bacteriochlorin bears twogeminal dimethyl groups to lock in the bacteriochlorin hydrogenationlevel, two β-substituents, and zero (13) or one (12) methoxy group in ameso position. The self-condensation of dihydrodipyrrin-acetal 11proceeds under mild acid catalysis at room temperature withoutrequirement for an oxidant and affords the free base bacteriochlorins in˜40% yield (12+13). Workable quantities (10-100 mg) of thebacteriochlorins can be readily prepared. The bacteriochlorins exhibitcharacteristic absorption and fluorescence properties. Thebacteriochlorins are resistant to dehydrogenation and are stable to avariety of reaction conditions. This approach should provide readyaccess to bacteriochlorins bearing a variety of substituents, anessential feature for fundamental studies and use in diverseapplications.

EXAMPLE 2 Bacteriochlorins via a Dihydrodipyrrin-Carboxaldehyde

Recently and as described above, we developed a concise synthetic routeto stable bacteriochlorins (Kim, H.-J.; Lindsey, J. S. J. Org. Chem.2005, 70, 5475-5486). The synthesis employs the self-condensation of adihydrodipyrrin-acetal. Here we describe a new synthetic route tobacteriochlorins. The new route involves oxidation of a dihydrodipyrrinto give the corresponding dihydrodipyrrin-aldehyde, which undergoesself-condensation to give free base bacteriochlorins (H-BC and anunidentified-BC) and a tetradehydrocorrin-aldehyde. The new route can beused as an alternative route for bacteriochlorin synthesis. Use of thealdehyde rather than the acetal affords a number of advantages.

Results and Discussion

1. Approach. In our previous work, we prepared a number ofhydrodipyrrins and examined the reactivity of those hydrodipyrrins tofind synthetic routes to stable bacteriochlorins. Three prototypicaltarget hydrodipyrrin structures are shown in Chart 6.

Each hydrodipyrrin contains a pyrrole ring and a pyrroline ring. We wereable to prepare A but were not able to obtain B and C. We eventuallyfound that dihydrodipyrrin-acetal A underwent self-condensation to givebacteriochlorins. Indeed, when R is a p-tolyl group, thedihydrodipyrrin-acetal (1) gives two bacteriochlorins (H-BC, MeO-BC) anda tetradehydrocorrin (TDC). The yield of each macrocycle was reasonable(yields of 30-49% for H-BC and MeO-BC and 66% for TDC; optimized for therespective macrocycle) and can be controlled by the reaction conditions.The three macrocycles are shown in Scheme 6.

We are interested in achieving new route and better synthetic control ofbacteriochlorin formation because the reaction gave three differentmacrocycles. Moreover, the reaction mechanism of self-condensation ofthe acetal is not clear. We turned to two new targets, aldehydederivatives of a dihydrodipyrrin or a tetrahydrodipyrrin (B and C); suchintermediates may furnish a superior synthetic method and alsofacilitate probing the bacteriochlorin-forming process. However, we wereunable to prepare either B or C previously.

The new synthetic route to the targets B and C is summarized in Scheme7. Routes to both derivatives were inspired by the work of Jacobi et al(Org. Lett. 2001, 3, 831-834). They converted the α-methyl group of apyrroline unit to the formyl group via oxidation with SeO₂. Accordingly,we focused on the synthesis of dihydrodipyrrin-aldehyde derivatives andtheir self-condensation.

2. Synthesis of Dihydrodipyrrin-Aldehyde (6). The synthesis ofβ-substituted dihydrodipyrrin 5 was initiated in a manner analogous tothat of previous dihydrodipyrrins (Balasubramanian, T. et al., J. Org.Chem. 2000, 65, 7919-7929). Treatment of nitroethyl pyrrole 3 withmesityl oxide containing DBU gave γ-nitrohexanone 4 in 74% yield viaMichael addition, as reported recently (Ptaszek, M. et al., Org. ProcessRes. Dev. 2005, in press). Subsequent treatment of 4 with NaOMe followedby a buffered TiCl₃ solution afforded dihydrodiyrrin 5 as a yellow solidin 25% yield. Oxidation of dihydrodipyrrin 5 with SeO₂ gave thecorresponding dihydrodipyrrin-aldehyde 6 in 47% yield (Scheme 8). Thelow yield of the oxidation is attributed to the instability of thealdehyde 6. On the other hand, all attempts to carry out a similaroxidation of tetrahydrodipyrrin 2→C were unsuccessful.

3. Effect of Reactant and Acid Concentrations. For immediate comparisonof the self-condensation between the dihydrodipyrrin-acetal and thedihydrodipyrrin-aldehyde, microscale experiments were performed underfour different acid concentrations. The concentration ofdihydrodipyrrin-aldehyde 6 was 4.7 mM and the concentration of BF₃.OEt₂varied from 0.47 to 47 mM. Each reaction was monitored over time (18 h).Two bacteriochlorins (H-BC and an unidentified-BC, though expected to bea hydroxybacteriochlorin or a oxo-bacteriochlorin) and atetradehydrocorrin-aldehyde (TDC-CHO) were detected by absorptionspectroscopy and LD-MS (Scheme 9). The collected data for H-BC wereconsistent with the same bacteriochlorin obtained from the correspondingacetal. LD-MS analysis of TDC-CHO gave a molecule ion peak (m/z=566.2)consistent with the proposed structure; moreover, the absorptionspectrum was quite similar with that of TDC (not shown).

The total yield of bacteriochlorins (but not TDC-CHO) was determinedspectroscopically from the crude mixture. Each reaction mixture was thenseparated chromatographically to determine the isolated yield of thetetradehydrocorrin-aldehyde TDC-CHO (Table 1). The highest spectroscopicyield of the bacteriochlorins was 11% at 47 mM of BF₃.OEt₂ (entry 4). Itis noteworthy that the yield of the sum of the bacteriochlorins (entry4) decreased slowly as the reaction proceeded. The highest yield ofTDC-CHO (˜4%) was obtained at 2.3 mM of BF₃.OEt₂ (entry 2). The yield ofTDC was highest at relatively low acid concentration and was notobserved at the highest acid concentration (47 mM).

TABLE 1 Spectroscopic Yields of Hydroporphyrins from MicroscaleReactions^(a) [BF₃•OEt₂], Yield (Sum of H-BC and TDC-CHO Entry [6], mMmM an unidentified BC) yield^(d) 1^(b) 4.7 0.47 0.2%^(c) (5 h)^(e) 1%(18 h) 2^(b) 4.7 2.3 0.7%^(c) (1 h)^(e) 4% (18 h) 3^(b) 4.7 4.7 0.8%^(c)(5 h)^(e) 1% (18 h) 4^(b) 4.7 47  11%^(c) (1 h)^(e) — ^(a)Spectroscopicyields were determined assuming ϵ_(Qy) = 120,000 M⁻¹cm⁻¹ forbacteriochlorins and ϵ_(363 nm) = 24,000 M⁻¹cm⁻¹. ^(b)The reaction wasperformed using 5.1 μmol of 6. ^(c)Highest yield was obtained from crudemixture. ^(d)Yield was obtained from isolated fraction. ^(e)The datapoints were taken at 1, 5, and 18 h and the reaction time was taken athighest yield.

4. Synthesis of a Bacteriochlorin (H-BC). The reaction was designed tofavor H-BC. The reaction conditions employed for the self-condensationof dihydrodipyrrin-aldehyde 6 were similar to those from the previouscondensation of dihydrodipyrrin-acetal 1. The conditions entailed ˜3.7mM of aldehyde 6 in CH₃CN containing 79 mM of BF₃.OEt₂ at roomtemperature. Samples were removed (50 μL) three times (0.5, 2, 17 h),neutralized with TEA, and examined by absorption spectroscopy. Yieldswere calculated on the basis that the bacteriochlorin has ε_(Qy)=130,000M⁻¹cm⁻¹. The spectroscopic yields of the bacteriochlorin in the crudemixture were ˜3% (0.5 h), ˜6% (2 h), and ˜6% (17 h) respectively. Theisolated yield of H-BC was ˜5% (Scheme 9).

Experimental Section

General. ¹H NMR (400 MHz) spectra were collected at room temperature inCDCl₃. Column chromatography was performed with flash silica or alumina(80-200 mesh). Bacteriochlorin was analyzed in neat form by laserdesorption mass spectrometry (LD-MS) in the absence of a matrix.Compounds 1 (Taniguchi, M. et al., J. Org. Chem. 2001, 66, 7342-7354)and 3 (Kim, H.-J.; Lindsey, J. S. J. Org. Chem. 2005, 70, 5475-5486)were prepared as described in literature.

6-[3-(4-Methylphenyl)pyrrol-2-yl]-4,4-dimethyl-5-nitro-2-hexanone (4).Following a standard procedure (Ptaszek et al., supra), a mixture of 3(460 mg, 2.00 mmol) and mesityl oxide (458 μL, 4.00 mmol) in CH₃CN (4.0mL) was treated with DBU (897 μL, 6.00 mmol). The reaction mixture wasstirred for 24 h at room temperature, diluted with ethyl acetate (15.0mL) and washed with water and brine. The organic layer was dried(Na₂SO₄) and concentrated. Excess mesityl oxide was removed under highvacuum. The resulting oil was chromatographed [ethyl acetate/hexanes(1:2)] to afford a light brown oil (486 mg, 74%): ¹H NMR δ 1.08 (s, 3H),1.19 (s, 3H), 2.10 (s, 3H), 2.37 (s, 3H), 2.37, 2.55 (AB, ²J=17.6 Hz,2H), 3.21 (ABX, ³J=2.6 Hz, ²J=15.6 Hz, 1H), 3.38 (ABX, ³J=11.6 Hz,²J=15.6 Hz, 1H), 5.18 (ABX, ³J=2.6 Hz, ³J=11.6 Hz, 1H), 6.22-6.24 (m,1H), 6.67-6.69 (m, 1H), 7.20 (d, J=8.0 Hz, 2H), 7.24 (d, J=8.0 Hz, 2H),8.05-8.20 (br, 1H).

1,3,3-Trimethyl-7-(4-methylphenyl)-2,3-dihydrodipyrrin (5). Following astandard procedure (see, e.g., Strachan, J.-P. et al., J. Org. Chem.2000, 65, 3160-3172; Strachan, J.-P. et al., J. Org. Chem. 2001, 66,642), a solution of 4 (256 mg, 0.780 mmol) in anhydrous THF (7.8 mL)under argon was treated with NaOMe (210 mg, 3.90 mmol). The mixture wasargon bubbled for 10 min and was stirred for 1 h at room temperature(first flask). In a second flask, TiCl₃ [8.6 wt % TiCl₃ in 28 wt % HCl(d=1.2), 5.83 mL, 3.90 mmol, 5.0 mol equiv] and H₂O (31 mL) werecombined. The solution was argon bubbled for 10 min NH₄OAc (22.9 g, 297mmol) was slowly added to buffer the solution to pH 6.0; and then THF(2.2 mL) was added under argon bubbling (˜20 min). The solution in thefirst flask containing the nitronate anion of 4 was transferred via acannula to the buffered TiCl₃ solution in the second flask. Theresulting mixture was stirred at room temperature for 6 h under argon.Then the mixture was slowly poured to a stirring solution of saturatedaqueous NaHCO₃ (120 mL) and ethyl acetate (40 mL). After 10 min, themixture was extracted with ethyl acetate. The combined organic layerswere washed with water and then dried (NaSO₄). The solvent was removedunder reduced pressure at room temperature. The crude product was passedthrough a short column [alumina, hexanes/ethyl acetate (2:1)] to afforda yellow solid (55 mg, 25%): ¹H NMR δ 1.18 (s, 6H), 2.21 (s, 3H), 2.38(s, 3H), 2.51 (s, 2H), 5.97 (s, 1H), 6.27-6.28 (m, 1H), 6.84-6.86 (m,1H), 7.21 (d, J=8.0 Hz, 2H), 7.36 (d, J=8.0 Hz, 2H), 10.99-11.10 (br,1H).

1-Formyl-3,3-dimethyl-7-(4-methylphenyl)-2,3-dihydrodipyrrin (6).Following a general procedure (Jacobi, P. A. et al., Org. Lett. 2001, 3,831-834), a solution of 5 (45 mg, 0.16 mmol) in 1,4-dioxane (3.2 mL) wastreated with SeO₂ (27 mg, 0.24 mmol) under argon. The mixture wasstirred for 1.5 h at room temperature. The reaction mixture was treatedwith saturated NaHCO₃ (4.0 mL) and extracted with ethyl acetate. Theorganic extract was washed with water, dried (Na₂SO₄), andchromatographed [silica, ethyl acetate/hexanes (1:3)] to give a darkorange solid (22 mg, 47%): ¹H NMR δ 1.22 (s, 6H), 2.41 (s, 3H), 2.72 (s,2H), 6.34-6.35 (m, 1H), 6.38 (s, 1H), 7.00-7.02 (m, 1H), 7.25 (d, J=8.0Hz, 2H), 7.35 (d, J=8.0 Hz, 2H), 10.0 (s, 1H), 10.71-10.82 (br, 1H).FAB-MS obsd 293.1654, calcd 293.1654 [(M+H)⁺, M=C₁₉H₂₀N₂O].

8,8,18,18-Tetramethyl-2,12-bis(4-methylphenyl)bacteriochlorin (7, H-BC).A solution of 6 (˜1 mg, 3.4 μmol) in CH₃CN (1.0 mL) was treated withBF₃.OEt₂ (10 μL, 79 μmol). The reaction mixture was stirred at roomtemperature without deaeration for 23 h. TEA (12 μL, 86 μmol) was addedto the reaction mixture. The reaction mixture was concentrated and theresidue was chromatographed [silica, CH₂Cl₂/hexanes (1:1)] to give agreen solid (H-BC, ˜0.05 mg, 5%). The product co-chromatographed[silica, hexanes/CH₂Cl₂ (1:1), R_(f)=0.77] with an authentic sample ofH-BC prepared from the dihydrodipyrrin-acetal 1. The absorption spectrumand LD-MS data were consistent with previously reported values: λ_(abs)(CH₂Cl₂)/nm 350, 373, 498, 737; LD-MS obsd 549.5, calcd 550.31(C₃₈H₃₈N₄).

The foregoing is illustrative of the present invention, and is not to beconstrued as limiting thereof. The invention is defined by the followingclaims, with equivalents of the claims to be included therein.

1. (canceled)
 2. In a method of detecting cells or particles by flow cytometry, wherein said cells or particles are labelled with a detectable luminescent compound, the improvement comprising utilizing a bacteriochlorin as the luminescent compound, wherein said bacteriochlorin is a compound of Formula I:

wherein: M is a metal or is absent; X is NH; R¹ and R² are each independently selected from the group consisting of H, alkyl, aryl, alkoxy, halo, mercapto, cyano, hydroxyl, nitro, acyl, alkylthio, alkylamino, acyloxy, linking groups, and —C(O)NR_(a)R_(b) where R_(a) and R_(b) are each independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl and aryl; R³ and R⁴ are each independently selected from the group consisting of alkyl, cycloalkyl, aryl, arylalkyl, and linking groups; R⁵ is selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, alkoxy, halo, cyano, nitro, acyl, alkylthio, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, linking groups, and —C(O)NR_(a)R_(b) where R_(a) and R_(b) are each independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl and aryl; R⁶ and R⁷ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, halo, cyano, nitro, acyl, alkoxy, alkylthio, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, linking groups, —C(O)NR_(a)R_(b) where R_(a) and R_(b) are each independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl and aryl, —C(O)OR_(c) where R_(c) is selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl and aryl, and C(O)OH; and R⁸ is selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, arylalkyl, arylalkenyl, arylalkynyl, halo, cyano, nitro, acyl, alkoxy, alkylthio, alkylamino, arylalkylamino, disubstituted amino, acylamino, acyloxy, linking groups, and —C(O)NR_(a)R_(b) where R_(a) and R_(b) are each independently selected from the group consisting of alkyl, cycloalkyl, alkenyl, alkynyl and aryl; or R¹ and R² together are ═O or spiroalkyl; or R³ and R⁴ together are ═O or spiroalkyl; subject to the proviso that (i) neither R¹ nor R² is H, or (ii) neither R³ nor R⁴ is H; and further subject to the proviso that, when X is NH: R¹ is not cycloalkyl; or R² is not methyl; or R⁵ is not H; or R⁶ is not H; or R⁷ is not methyl.
 3. The method of claim 2, wherein R¹ is not cycloalkyl.
 4. The method of claim 2, wherein R² is not methyl.
 5. The method of claim 2, wherein R⁵ is not H.
 6. The method of claim 2, wherein R⁶ is not H.
 7. The method of claim 2, wherein R⁷ is not methyl.
 8. The method of claim 2, wherein M is present and is selected from the group consisting of Pd, Pt, Mg, Zn, Al, Ga, In, Sn, Cu, Ni, and Au.
 9. The method of claim 2, wherein at least one of R¹ through R⁸ is a linking group.
 10. The method of claim 2, wherein at least one of R⁶, R⁷, or R⁸ is a linking group.
 11. The method of claim 2, wherein the compound is coupled to a hydrophilic group.
 12. The method of claim 2, wherein the compound is coupled to a hydrophilic group at at least one of said R⁶, R⁷, or R⁸ positions.
 13. The method of claim 2, wherein the compound is coupled to a targeting agent.
 14. The method of claim 2, wherein the compound is coupled to a targeting agent at at least one of said R⁶, R⁷, or R⁸ positions.
 15. The method of claim 2, wherein the compound is to an antibody.
 16. The method of claim 2, wherein the compound is coupled to an antibody at at least one of said R⁶, R⁷, or R⁸ positions.
 17. The method of claim 2, wherein the compound is coupled to a protein or peptide.
 18. The method of claim 2, wherein the compound is coupled to a protein or peptide at at least one of said R⁶, R⁷, or R⁸ positions.
 19. The method of claim 2, wherein the compound is coupled to a nucleic acid.
 20. The method of claim 2, wherein the compound is coupled to a nucleic acid at at least one of said R⁶, R⁷, or R⁸ positions. 